Allergenic proteins and peptides from dog dander and uses therefor

ABSTRACT

Isolated nucleic acids encoding allergens of  Canis familiaris,  Can f I or Can f II, are disclosed. A cDNA encoding a peptide having a Can f I activity and a predicted molecular weight of about 19,200 daltons is also described. A cDNA encoding a peptide having Can f II activity and a predicted molecular weight of about 18,200 daltons is also disclosed. The nucleic acids can be used as probes to detect the presence of Can f I or Can f II nucleic acid in a sample or for the recombinant production of peptides having a Can f I or Can f II activity. Peptides having a Can f I or Can f II activity can be used in compositions suitable for pharmaceutical administration or methods of diagnosing sensitivity to dog dander.

BACKGROUND OF THE INVENTION

Approximately 10% of the population become hypersensitized (allergic)upon exposure to antigens from a variety of environmental sources. Thoseantigens that induce immediate and/or delayed types of hypersensitivityare known as allergens (King, T. P., (1976) Adv. Immunol., 23: 77-105.These include products of grasses, trees, weeds, animal dander, insects,food, drugs, and chemicals. Genetic predisposition of an individual isbelieved to play a role in the development of immediate allergenicresponses (Young, R. P. et al., (1990) Clin. Sci., 79: 19a) such asatopy and anaphylaxis whose symptoms include hay fever, asthma, andhives.

The antibodies involved in atopic allergy belong primarily to the IgEclass of immunoglobulins. IgE binds to basophils, mast cells anddendritic cells via a specific, high-affinity receptor FcεRI (Kinet, J.P., (1990) Curr. Opin. Immunol., 2: 499-505). Upon combination of anallergen acting as a ligand with its cognate receptor IgE, FcεRI boundto the IgE may be cross-linked on the cell surface, resulting inphysiological manifestations of the IgE—allergen interaction. Thesephysiological effects include the release of, among other substances,histamine, serotonin, heparin, chemotactic factor(s) for eosinophilicleukocytes and/or leukotrienes C4, D4, and E4, which cause prolongedconstriction of bronchial smooth muscle cells (Hood, L. E. et al.,Immunology (2nd ed.), The Benjamin/Cumming Publishing Co., Inc. (1984)).Hence, the ultimate consequence of the interaction of an allergen withIgE are allergic symptoms triggered by release of the aforementionedmediators. Such symptoms may be systemic or local in nature, dependingon the route of entry of the antigen and the pattern of deposition ofIgE on mast cells or basophils. Local manifestations generally occur onepithelial surfaces at the site of entry of the allergen. Systemiceffects can induce anaphylaxis (anaphylactic shock) which results fromIgE-basophil response to circulating (intravascular) antigen.

The pet dog (Canis familiaris) is kept in households the world over. Inhouses and public schools where dogs have been kept on a regular basis,dog dander allergens can be detected in dust samples (Wood, R. A. etal., (1988) Am Rev Respir. Dis., 137: 358-363, and Dybendal, T. et al.,(1989) Allergy, 44: 401-411). The prevalence of allergy to dogs asassessed by skin prick test is approximately 15% (Haahtela, T. et al.,(1981) Allergy, 36: 251-256, and de Groot, H. et al., (1991) J. AllergyClin. Immunol., 87: 1056-1065). In one study, sensitivity to dogallergen(s) was detected in 40% of asthmatic children, even though dogswere not kept as pets in their homes (Vanto, T. and Koivikko, A., (1983)Acta Paediatr Scand., 72: 571-575).

Treatment of patients with dog allergy by administration of dog danderextracts has not proven to be as efficacious as treatment of catallergic patients with cat dander extracts (Hedlin, G. et al., (1991) J.Allergy Clin Immunol., 87: 955-964). As with any desensitization schemeinvolving injection of increasing doses of allergen(s), there are thedrawbacks of potential anaphylaxis during treatment, and the possiblenecessity of continuing therapy over a period of several years to buildup sufficient tolerance that results in significant diminution ofclinical symptoms.

Dog hair and dander extracts are complex mixtures containing a number ofallergenic proteins. (Loewenstein, H et al., (1982) Proceedings 11thInternational Congress of Allergology and Clinical Immunology, London,pp 545-548; Uchlin, T et al., (1984) Allergy, 39: 125-133; Yman, L. etal., (1984) Int. Arch. Allergy Appl. Immunol. U, 44: 358-368; Spitzauer,S. et al., (1993) Int. Arch. Allergy Immunol., 100: 60-67). Twoallergens present in dog hair/dander have been purified usingimmunoaffinity chromatography. A major allergen from dog, Can f I(Nomenclature according to the criteria of the IUIS (Marsh, D. G. etal., (1988) Clin. Allergy, 18: 201-209; Ag13 according to originalnomenclature), has been partially purified by two groups (Schou, C. etal., (1991) Clin. and Exp. Allergy, 21: 321-328 and de Groot et al.,supra). Both groups, partially purified Can f I was established as anallergen by CRIE analysis (Ford A. W. et al., (1989) Clin. Exp. Allergy,19: 183-190), and then rabbits or Balb/b mice were immunized to obtainpolyclonal or monoclonal antibodies against the allergen, respectively.Immunoaffinity purified Can f I (˜25 kD in molecular weight, with aminor component ˜18 kD) which elicited a high frequency of positive skinprick tests among dog allergic patients was able to deplete 50-70% ofIgE binding to dog dander extracts in RAST (radioallergosorbent test)analysis. While de Groot et al, did not attempt to determine any aminoacid sequence of Can f I, Schou et al, found the amino terminus of theirimmunoaffinity purified Can f I was blocked. Hence, no amino acidsequence of Can f I is presently in the public domain.

The presence of a second (minor) allergen in dog extract was detected bybinding of IgE antibodies to dog dander/hair extracts by several groups(de Groot et al., supra, Schou, C. et al., supra and Spitzauer et al.,(1993) Int. Arch. Allergy Immunol., 100: 60-67). The molecular weight ofa minor allergen was reported to be 18 kD (Schou et al., supra), 19 kD(Spitzauer et al., supra) and 27 kd (de Groot et el., supra). It isdifficult however to correlate these results since only one group (deGroot et al., supra) affinity purified an allergen designated Can f II(originally named Dog 2 allergen). Can f II was purified from dog danderextracts in a manner analogous to Can f I using monoclonal antibodiesgenerated to a second allergen present in extracts (de Groot et al.,supra). Molecular weight of Can f II reported by this group as ˜27 kDwas later verified to be ˜24 kD (Aalberse, R. C. personalcommunication). Purified Can f II allergen was found to react with IgEof only 66% of dog allergic patients. In RAST analysis, Can f IIallergen was able to compete with 23% of the IgE directed against dogdander extract. The amino acid sequence of Can f II has not beenpreviously determined.

Many patients with sensitivity to dog dander allergens are treatedcurrently by administration of small, gradually increasing doses of dogdander extracts. Use of these extracts has multiple drawbacks, includingpotential anaphylaxis during treatment and the necessity of continuingtherapy, often for a period of several years to build up sufficienttolerance and significant diminution of clinical symptoms. The abilityto substitute compositions of at least the major dog dander allergens,such as Can f I and Can f II, would overcome several of these drawbacks.Thus, a source of pure allergen that could be provided in quantity foruse as a diagnostic or therapeutic reagent and therapeutic methods thatwould overcome the drawbacks associated with dog dander extracts arehighly desirable.

SUMMARY OF THE INVENTION

This invention provides isolated nucleic acids encoding peptides havingat least one biological activity of Can f I or Can f II, proteinallergens of the species Canis familiaris. Preferred nucleic acids arecDNAs having a nucleotide sequence shown in FIG. 5 (SEQ ID NO: 1)(Can fI) and FIG. 18 (SEQ ID NO: 67)(Can f II). The invention also pertains topeptides encoded by all or a portion of such cDNAs (SEQ ID NO:1 and SEQID NO: 67) and having at least one biological activity of Can f I or Canf II. Also contemplated are isolated nucleic acids which hybridize underhigh stringency conditions (e.g., equivalent to 20-27° C. below Tm and1M NaCl) to a nucleic acid having a nucleotide sequence shown in FIG. 5(SEQ ID NO: 1) or FIG. 18 (SEQ ID NO: 67) or which encodes a peptidecomprising all or a portion of an amino acid sequence of FIG. 5 (SEQ IDNO: 2)(Can f I) or FIG. 18 (SEQ ID NO: 68)(Can f II). Nucleic acidswhich encode peptides having an activity of Can f I or Can f II andhaving at least 50% homology with a sequence shown in FIG. 5 (SEQ ID NO:2)(Can f I) or FIG. 18 (SEQ ID NO: 68)(Can f II) are also featured.Peptides having a Can f I or Can f II activity produced by recombinantexpression of a nucleic acid of the invention, and peptides having a Canf I or Can f II activity prepared by chemical synthesis are alsofeatured by this invention. Preferred peptides have the ability toinduce a T cell response, which may include T cell stimulation (measuredby, for example, T cell proliferation or cytokine secretion) or T cellnonresponsiveness (i.e., contact with the peptide or a complex of thepeptide with an MHC molecule of an antigen presenting cell induces the Tcell to become unresponsive to stimulatory signals or incapable ofproliferation). Other preferred peptides, either apart from or inaddition to the ability to induce a T cell response, have the ability tobind the dog dander specific IgE of dog dander-allergic subjects. Suchpeptides are useful in diagnosing sensitivity to dog dander in asubject. Still other peptides, either apart from or in addition to theability to induce a T cell response, have a significantly reduced ornegligible ability to bind dog dander-allergic IgE. Such peptides areparticularly useful as therapeutic agents.

Other preferred peptides comprise an amino acid sequence shown in FIG. 5(SEQ ID NO: 2)(Can f I) or FIG. 18 (SEQ ID NO: 68)(Can f II). In oneembodiment, peptides having a Can f I or Can f II activity andcomprising a portion of the amino acid sequence of FIG. 5 (SEQ ID NO: 2)or FIG. 18 (SEQ ID NO: 68) are at least about 8-30 amino acids inlength, preferably about 10-20 amino acids in length, and mostpreferably about 10-16 amino acids in length.

Another aspect of the invention features antibodies specificallyreactive with a peptide having a Can f I or Can f II activity. A peptidehaving an activity of Can f I or Can f II can be used in compositionssuitable for pharmaceutical administration. For example, suchcompositions can be used in a manner similar to dog dander extracts totreat or prevent allergic reactions to dog dander in a subject. Nucleicacids of the invention and peptides having an activity of Can f I or Canf II can be also used for diagnosing sensitivity in a subject to a dogdander.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows degenerate primer pairs based on residues 9-15 and 30-37 ofthe mature Can f I protein used in the MOPAC technique of PCRamplification. Two internal degenerate oligonucleotide probes based onCan f I protein residues 17-24 (Dog Probe 1) and 88-94 (Dog Probe 2) areshown.

FIG. 2 shows oligonucleotide primers used in a RACE PCR protocol toobtain the 3′ portion of the Can f I cDNA. A degenerate oligonucleotideprobe (Dog Probe 4) is also shown.

FIG. 3 shows primers used in an anchored PCR technique to determine the5′ end of the Can f I cDNA. A degenerate oligonucleotide probe, DogProbe 0, based on residues 9-17 of the Can f protein is shown.

FIG. 4 is a schematic representation of a PCR sequencing strategy usedto obtain the sequence of the mature Can f I protein from both strandsof amplified cDNA.

FIG. 5 is the cDNA sequence and deduced amino acid sequence of Can f I.

FIG. 6 is a schematic representation of the strategy used to express Canf I recombinant protein in bacteria.

FIG. 7 is a schematic representation of the strategy used to express Canf I recombinant protein in a mammalian cell using the pJ7L expressionvector.

FIG. 8 is a schematic representation of the strategy used to insert aHis6 reporter group at the carboxy terminus of the recombinant Can f Iprotein to aid purification of the protein.

FIG. 9 shows the alignment of three partial 3′ Can f I cDNA sequences(Can f I, 2Can f I and 3Can f I). An (*) indicates that the position inthe alignment is perfectly conserved and a (·) indicates that theposition is well conserved. A (−) was inserted where necessary forpurposes of alignment.

FIG. 10 is a graphic representation depicting the response of T celllines from patients primed in vitro with recombinant Can f I (rCan f I)and analyzed for response to rCan f I and various peptides derived fromCan f I by positivity index (% of patients who positively respondedmultiplied by the mean stimulation index).

FIG. 11 is a graphic representation of a direct binding assay of IgEfrom a single dog allergic patient to bacterially expressed recombinantCan f I.

FIG. 12 shows Western blot analysis of four protein preparations (Lane1: dog hair extract; Lane 2: dog saliva; Lane 3: bacterially expressedrecombinant Can f I; and Lane 4: recombinant Can f I expressed in amammalian cell culture system) probed with plasma from a dog allergicpatient (#901) or with plasma from a negative control patient (#250).

FIG. 13 shows the design of primers based on a partial amino acidsequence of mature Can f II and the sequence strategy for Can f II. ( )denotes residues which were not determined.

FIG. 14 is a schematic representation of the strategy used to clone Canf II cDNA.

FIG. 15 is a schematic representation of the strategy used to clone the5′ (A) and 3′ (B) portions of Can f II cDNA flanking the sequenceencoding a portion of the amino acid sequence (shaded) of native Can fII.

FIG. 16 is the nucleotide sequence of primers used in cloning Can f IIcDNA.

FIG. 17 shows the sequence strategy used to determine the nucleotidesequence of the Can f II cDNA clones 1a, 1c and 1j. The figure depictsinserts of the cDNA clones 1a (793 bp), 1c (791 bp) and 1j (774 bp). Thehatched bars represent coding sequence. The triangles indicate theposition of an initiator methionine codon (ATG); the codon specifyingthe N-terminal amino acid residue of the mature protein (START); theposition of a termination codon (STOP); and the position of apolyadenylation signal (As). The arrows indicate the extent anddirection of the sequencing reactions.

FIG. 18 is the cDNA sequence and deduced amino acid sequence (from Clone1c) of Can f II.

FIG. 19 is a comparison of the deduced amino acid sequence of Can f IIbased on the cDNA sequence of clone 1c and a portion of the native Can fII determined by protein sequencing of the N-terminus. The amino acidresidues of a signal peptide are numbered −19 to −1.

FIG. 20 shows northern analysis of mRNA of different dog tissues. Totalcellular RNA (25 mg) from dog tongue epithelial tissue, parotid salivarygland, skin, mandibular and submaxiliary glands, liver and spleen wassubjected to Northern analysis using Can f II cDNA as a probe. Theposition of RNA markers are indicated in kilobases (kb).

FIG. 21 is a comparison of the amino acid sequence of Can f II withhomologus proteins MUP 6 Mouse and Rat A2U. The alignment was made withGeneWorks program. The signal sequences are underlined. Amino acidresidues which are identical in all three proteins are boxed.

FIGS. 22A-C are graphic representations of a direct binding assays ofhuman IgE binding to native Can f II and recombinant Can f II.

FIG. 23 is the nucleotide consensus sequence among cDNA clones 1a, 1cand 1j encoding partial or full length Can f II.

FIG. 24 is a graphic representation depicting the response of T celllines from 12 patients primed in vitro with recombinant Can f I (rCan fI) and analyzed for response to rCan f I and various peptides derivedfrom Can f I by stimulation index. A stimulation index equal to orgreater than two times the background is considered “positive.”

FIG. 25 is a graphic representation depicting the response of T celllines from 12 patients primed in vitro with recombinant Can f I (rCan fI) and analyzed for response to rCan f I and various peptides derivedfrom Can f I by mean stimulation index for the group of patients withpositive responses to the peptides.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to isolated nucleic acids encoding peptideshaving at least one biological activity of Can f I or Can f II,allergens of the species Canis familiaris. Preferably, the nucleic acidis a cDNA comprising a nucleotide sequence shown in FIG. 5 (SEQ ID NO:1)(Can f I) or FIG. 18 (SEQ ID NO:67)(Can f II).

The cDNA shown in FIG. 5 (SEQ ID NO:1) encodes a Can f I peptide whichincludes a 26 amino acid leader sequence encoded by base 1 through base78. This leader sequence is not found in the mature Can f I protein,which is encoded by bases 79 through 525. The deduced amino acidsequence of Can f I based on this cDNA is also shown in FIG. 5 (SEQ IDNO: 2). The cDNA encodes a mature peptide having a predicted molecularweight of 19.2 kDa, with a pI of 5.53 and a single potential N-linkedglycosylation site. A culture of E. coli transfected with an expressionvector containing the cDNA encoding Can f I was deposited under theBudapest Treaty with the American Type Culture Collection on Dec. 22,1992 and assigned accession number 69167.

The cDNA shown in FIG. 18 (SEQ ID NO: 67) encodes a Can f II peptidewhich includes a 19 amino acid leader sequence encoded by base 195through base 251. This leader sequence is not found in the mature Can fII protein, which is encoded by bases 252 through 734. The deduced aminoacid sequence of Can f II based on this cDNA is shown in FIG. 18 (SEQ IDNO: 68). The cDNA encodes a Can f II peptide having a predictedmolecular weight of 18.229 kDa, with a pI of 4.54 for a maturerecombinant Can f II protein and a pI of 4.44 for a full length(including signal sequence), recombinant Can f II protein, and a singlepotential N-linked glycosylation site. N-linked glycosylation mayincrease the molecular weight of the peptide and may alter the pI of themature protein. A culture of E. coli transfected with an expressionvector containing the cDNA encoding Can f II was deposited under theBudapest Treaty with the American Type Culture Collection (ATCC), P.O.Box 1549, Manassas, Va. 20108, USA on Dec. 29, 1993 and assignedaccession number 69167.

Accordingly, one aspect of this invention pertains to isolated nucleicacids comprising nucleotide sequences encoding Can f I or Can f II,fragments thereof encoding peptides having at least one biologicalactivity of Can f I or Can f II, and/or equivalents of such nucleicacids. The term nucleic acid as used herein is intended to include suchfragments and equivalents. The term equivalent is intended to includenucleotide sequences encoding functionally equivalent Can f I or Can fII proteins or functionally equivalent peptides having an activity ofCan f I or Can f II. As defined herein, a peptide having an activity ofCan f I or Can f II has at least one biological activity of the Can f Ior Can f II allergen. Equivalent nucleotide sequences will includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants, and will also include sequencesthat differ from the nucleotide sequence encoding Can f I or Can f IIshown in FIG. 5 (SEQ ID NO: 1) or FIG. 18 (SEQ ID NO: 67) due to thedegeneracy of the genetic code. Equivalents will also include nucleotidesequences that hybridize under stringent conditions (i.e., equivalent toabout 20-27° C. below melting temperature (T_(m)) and about 1M salt) tothe nucleotide sequence of Can f I shown in FIG. 5 (SEQ ID NO: 1) or Canf II shown in FIG. 18 (SEQ ID NO: 67).

Peptides referred to herein as having an activity of Can f I or Can f IIor having a Can f I or Can f II activity are defined herein as peptidesthat have an amino acid sequence corresponding to all or a portion ofthe amino acid sequence of Can f I or Can f II shown in FIG. 5 (SEQ IDNO: 2) or FIG. 18 (SEQ ID NO: 68) which peptide has at least onebiological activity of Can f I or Can f II. For example, a peptidehaving an activity of Can f I or Can f II may have the ability to inducea response in Can f I or Can f II restricted T cells such as stimulation(e.g., T cell proliferation or cytokine secretion) or to induce T cellnon-responsiveness. Alternatively, or additionally, a peptide having anactivity of Can f I or Can f II may have the ability to bind (to berecognized by) immunoglobulin E (IgE) antibodies of dog dander-allergicsubjects. Peptides which bind IgE are useful in methods of detectingallergic sensitivity to Can f I or Can f II in a subject. Peptides thatdo not bind IgE, or bind IgE to a lesser extent than a purified, nativeCan f I or Can f II protein binds IgE are particularly useful astherapeutic agents.

In one embodiment, the nucleic acid is a cDNA encoding a peptide havingan activity of Can f I or Can f II. Preferably, the nucleic acid is acDNA molecule comprising at least a portion of the nucleotide sequenceencoding Can f I or Can f II, shown in FIG. 5 (SEQ ID NO:1) and FIG. 18(SEQ ID NO: 67). A preferred portion of the cDNA molecules of FIG. 5 andFIG. 18 includes the coding region of the molecule.

In another embodiment, the nucleic acid of the invention encodes apeptide having an activity of Can f I or Can f II and comprising anamino acid sequence shown in FIG. 5 (SEQ ID NO:2) (Can f I) or FIG. 18(SEQ ID NO: 68) (Can f II). Preferred nucleic acids encode a peptidehaving a Can f I or Can f II activity and having at least about 50%homology, more preferably at least about 60% homology and mostpreferably at least about 70% homology with the sequence shown in FIG. 5(SEQ ID NO: 1) (Can f I) or FIG. 18 (SEQ ID NO: 67) (Can f II). Nucleicacids which encode peptides having a Can f I or Can f II activity andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence set forth inFIG. 5 (SEQ ID NO: 2) (Can f I) or FIG. 18 (SEQ ID NO: 68) (Can f II)are also within the scope of the invention. Homology refers to sequencesimilarity between two peptides having an activity of Can f I or Can fII or between two nucleic acid molecules. Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having all or a portion of an amino acid sequence shown inFIG. 5 (SEQ ID NO: 2) (Can f I) or FIG. 18 (SEQ ID NO: 68) (Can f II).Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° are known to those skilled in theart or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C.

Isolated nucleic acids encoding peptides having an activity of Can f Ior Can f II, as described herein, and having a sequence which differsfrom the nucleotide sequences shown in FIG. 5 (SEQ ID NO: 1) and FIG. 18(SEQ ID NO: 67) due to degeneracy in the genetic code are also withinthe scope of the invention. Such nucleic acids encode functionallyequivalent peptides (i.e., a peptide having an activity of Can f I orCan f II) but differ in sequence from the sequences of FIG. 5 and FIG.18 due to degeneracy in the genetic code. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the Can f I or Can f II protein. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequence of Can f I or Can f II will exist within the dogdander population. One skilled in the art will appreciate that thesevariations in one or more nucleotides (up to about 3-4% of thenucleotides) of the nucleic acids encoding peptides having an activityof Can f I or Can f II may exist among individual pet dogs due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention. Furthermore, there may be one or more isoforms or related,cross-reacting family members of Can f I or Can f II. Such isoforms orfamily members are defined as proteins related in function and aminoacid sequence to Can f I or Can f II, but encoded by genes at differentloci.

Fragments of the nucleic acid encoding Can f I or Can f II are alsowithin the scope of the invention. As used herein, a fragment of thenucleic acid encoding Can f I or Can f II refers to a nucleotidesequence having fewer nucleotides than the nucleotide sequence encodingthe entire amino acid sequence of Can f I or Can f II protein and whichencodes a peptide having an activity of Can f I or Can f II (i.e., apeptide having at least one biological activity of the Can f I or Can fII allergen) as defined herein.

Preferred nucleic acid fragments encode peptides of at least about 10amino acid residues in length, preferably about 10-20 amino acidresidues in length, and more preferably about 12-16 amino acid residuesin length. Nucleic acid fragments which encode peptides having a Can f Iactivity of at least about 30 amino acid residues in length, at leastabout 40 amino acid residues in length, at least about 60 amino acidresidues in length, at least about 80 amino acid residues in length, atleast about 100 amino acid residues in length, and at least about 140residues in length or more are also within the scope of this invention.Nucleic acid fragments which encode peptides having a Can f II activityof at least about 30 amino acid residues in length, at least about 40amino acid residues in length, at least about 60 amino acid residues inlength, at least about 80 amino acid residues in length, at least about100 amino acid residues in length, at least about 140 residues inlength, and at least about 160 amino acid residues in length or more arealso within the scope of this invention.

Nucleic acid fragments within the scope of the invention include thosecapable of hybridizing under high or low stringency conditions withnucleic acids from other animal species for use in screening protocolsto detect Can f I or Can f II or allergens that are cross-reactive withCan f I or Can f II. Generally, the nucleic acid encoding a peptidehaving an activity of Can f I or Can f II will be selected from thebases encoding the mature protein, however, in some instances it may bedesirable to select all or part of a peptide from the leader sequenceportion of the nucleic acids of the invention. Nucleic acids within thescope of the invention may also contain linker sequences, modifiedrestriction endonuclease sites and other sequences useful for molecularcloning, expression or purification of recombinant peptides having anactivity of Can f I or Can f II.

A nucleic acid encoding a peptide having an activity of Can f I or Can fII may be obtained from mRNA present in salivary glands or other organsof the pet dog Canis familiaris. It should also be possible to obtainnucleic acids encoding Can f I or Can f II from Canis familiaris genomicDNA. For example, the gene encoding Can f I or Can f II can be clonedfrom either a cDNA or a genomic library in accordance with protocolsherein described. A cDNA encoding Can f I or Can f II can be obtained byisolating total mRNA from Canis familiaris. Double stranded cDNAs canthen be prepared from the total mRNA. Subsequently, the cDNAs can beinserted into a suitable plasmid or bacteriophage vector using any oneof a number of known techniques. Genes encoding Can f I or Can f II canalso be cloned using established polymerase chain reaction techniques inaccordance with the nucleotide sequence information provided by theinvention. The nucleic acids of the invention can be DNA or RNA. Apreferred nucleic acid is a cDNA encoding Can f I or Can f II having thesequence depicted in FIG. 5 (SEQ ID NO:1) (Can f I) or FIG. 18 (SEQ IDNO: 67) (Can f II).

This invention also provides expression vectors containing a nucleicacid encoding a peptide having an activity of Can f I or Can f II,operably linked to at least one regulatory sequence. Operably linked isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the peptide having an activity of Can f I or Can f II.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transformed and/or the type of proteindesired to be expressed. In one embodiment, the expression vectorincludes a DNA encoding a peptide having an activity of Can f I or Can fII. Such expression vectors can be used to transfect cells to therebyproduce proteins or peptides, including fusion proteins or peptidesencoded by nucleic acids as described herein.

This invention further pertains to a host cell transfected to express apeptide having an activity of Can f I or Can f II. The host cell may beany procaryotic or eucaryotic cell. For example, a peptide having anactivity of Can f I or Can f II may be expressed in bacterial cells suchas E. coli, insect cells (baculovirus), yeast, or mammalian cells suchas Chinese hamster ovary cells (CHO). Other suitable host cells can befound in Goeddel, (1990) supra or known to those skilled in the art.

Expression in eucaryotic cells such as mammalian, yeast, or insect cellscan lead to partial or complete glycosylation and/or formation ofrelevant inter- or intra-chain disulfide bonds of recombinant protein.Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari. et al., (1987) Embo J., 6: 229-234), pMFa (Kurjan andHerskowitz, (1982) Cell, 30: 933-943), pJRY88 (Schultz et al., (1987)Gene, 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of proteins incultured insect cells (SF 9 cells) include the pAc series (Smith et al.,(1983) Mol. Cell Biol., 3: 2156-2165) and the pVL series (Lucklow, V.A., and Summers, M. D., (1989) Virology, 170: 31-39). Generally COScells (Gluzman, Y., (1981) Cell, 23: 175-182) are used in conjunctionwith such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl.Acad. Sci. USA, 84: 8573-8577) for transient amplification/expression inmammalian cells, while CHO (dhfr-Chinese Hamster Ovary) cells are usedwith vectors such as pMT2PC (Kaufman et al., (1987) EMBO J., 6: 187-195)for stable amplification/expression in mammalian cells. Vector DNA canbe introduced into mammalian cells via conventional techniques such ascalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, or electroporation. Suitable methodsfor transforming host cells can be found in Sambrook et al., (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

Expression in procaryotes is most often carried out in E. coli witheither fusion or non-fusion inducible expression vectors. Fusion vectorsusually add a number of NH2 terminal amino acids to the expressed targetgene. These NH2 terminal amino acids often are referred to as a reportergroup. Such reporter groups usually serve two purposes: 1) to increasethe solubility of the target recombinant protein; and 2) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the reportergroup and the target recombinant protein to enable separation of thetarget recombinant protein from the reporter group subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene, 69: 301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology, 185, Academic Press, San Diego,Calif. (1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET 11d relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated bycoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize recombinant Can f I or Can f II expression inE. coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology, 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy would be toalter the nucleic acid encoding the Can f I or Can f II protein to beinserted into an expression vector so that the individual codons foreach amino acid would be those preferentially utilized in highlyexpressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res., 20:2111-2118). Such alteration of nucleic acids of the invention can becarried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (See e.g., Itakura et al., U.S. Pat. No.4,598,049; Caruthers et al., U.S. Pat. No. 4,458,066; and Itakura, U.S.Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

The present invention further pertains to methods of producing peptidesthat have an activity of Can f I or Can f II. For example, a host celltransfected with a nucleic acid vector directing expression of anucleotide sequence encoding a peptide having an activity of Can f I orCan f II can be cultured under appropriate conditions to allowexpression of the peptide to occur. The peptide may be secreted andisolated from a mixture of cells and medium containing the peptidehaving an activity of Can f I or Can f II. Alternatively, the peptidemay be retained cytoplasmically and the cells harvested, lysed and theprotein isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The peptide having an activity of Can f I or Can f II can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for a peptidehaving an activity of Can f I or Can f II.

Another aspect of the invention pertains to isolated peptides having anactivity of Can f I or Can f II. A peptide having an activity of Can f Ior Can f II has at least one biological activity of the Can f I or Can fII allergen. For example, a peptide having an activity of Can f I or Canf II may have the ability to induce a response in Can f I or Can f IIspecific T cells such as stimulation (T cell proliferation or cytokinesecretion) or to induce T cell non-responsiveness. In one embodiment, apeptide having an activity of Can f I or Can f II stimulates T cells asevidenced by, for example, T cell proliferation or cytokine secretion.In another embodiment, peptides having a Can f I or Can f II activityinduce T cell non-responsiveness in which T cells are unresponsive to asubsequent challenge with a Can f I or Can f II peptide followingexposure to the peptide. In yet another embodiment, a peptide having aCan f I or Can f II activity has reduced IgE binding activity comparedto purified, native Can f I or Can f II protein. A peptide having anactivity of Can f I or Can f II may differ in amino acid sequence fromthe Can f I or Can f II sequence depicted in FIG. 5 (SEQ ID NO:2) (Can fI) or FIG. 18 (SEQ ID NO: 68) (Can f II) but such differences result ina modified protein which functions in the same or similar manner as anative Can f I or Can f II protein or which has the same or similarcharacteristics of a native Can f I or Can f II protein. Variousmodifications of the Can f I or Can f II protein to produce these andother functionally equivalent peptides are described in detail herein.The term peptide, as used herein, refers to peptides, proteins, andpolypeptides.

A peptide can be produced by modification of the amino acid sequence ofthe Can f I or Can f II protein shown in FIG. 5 (SEQ ID NO: 2) (Can f I)or FIG. 18 (SEQ ID NO: 68) (Can f II), such as a substitution, addition,or deletion of an amino acid residue which is not directly involved inthe function of the protein. Peptides of the invention can be at leastabout 10 amino acid residues in length, preferably about 10-20 aminoacid residues in length, and more preferably about 10-16 amino acidresidues in length. Peptides having an activity of Can f I or Can f IIand which are at least about 30 amino acid residues in length, at leastabout 40 amino acid residues in length, at least about 60 amino acidresidues in length, at least about 80 amino acid residues in length, andat least about 100 amino acid residues in length are also includedwithin the scope of this invention.

Another embodiment of the invention provides a substantially purepreparation of a peptide having an activity of Can f I or Can f II. Sucha preparation is substantially free of proteins and peptides with whichthe peptide naturally occurs (i.e., other canine peptides), either in acell or when secreted by a cell.

The term isolated as used herein refers to a nucleic acid or peptidethat is substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Such proteins or peptides arealso characterized as being free of all other dog dander proteins.Accordingly, an isolated peptide having an activity of Can f I or Can fII is produced recombinantly or synthetically and is substantially freeof cellular material and culture medium or substantially free ofchemical precursors or other chemicals and is substantially free of allother dog proteins. An isolated nucleic acid is also free of sequenceswhich naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the organism from which thenucleic acid is derived.

Peptides having an activity of Can f I or Can f II can be obtained, forexample, by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid of Can f I or Can f IIencoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, the Can fI or Can f II protein may be arbitrarily divided into fragments ofdesired length with no overlap of the fragments, or preferably dividedinto overlapping fragments of a desired length. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptides having a Can f I or Can f II activity (i.e., the abilityto induce a T cell response such as stimulation (proliferation, cytokinesecretion), nonresponsiveness, and/or has reduced IgE binding activity).

In one embodiment, peptides having an activity of Can f I or Can f IIcan be identified by the ability of the peptide to stimulate T cells orto induce T cell non-responsiveness. Peptides which stimulate T cells,as determined by, for example, T cell proliferation or cytokinesecretion are defined herein as comprising at least one T cell epitope.T cell epitopes are believed to be involved in initiation andperpetuation of the immune response to the protein allergen which isresponsible for the clinical symptoms of allergy. These T cell epitopesare thought to trigger early events at the level of the T helper cell bybinding to an appropriate HLA molecule on the surface of an antigenpresenting cell, thereby stimulating the T cell subpopulation with therelevant T cell receptor for the epitope. These events lead to T cellproliferation, lymphokine secretion, local inflammatory reactions,recruitment of additional immune cells to the site of antigen/T cellinteraction, and activation of the B cell cascade, leading to theproduction of antibodies. One isotype of these antibodies, IgE, isfundamentally important to the development of allergic symptoms and itsproduction is influenced early in the cascade of events at the level ofthe T helper cell, by the nature of the lymphokines secreted. A T cellepitope is the basic element, or smallest unit of recognition by a Tcell receptor, where the epitope comprises amino acids essential toreceptor recognition. Amino acid sequences which mimic those of the Tcell epitopes and which modify the allergic response to proteinallergens are within the scope of this invention.

Screening peptides for those which retain a Can f I or Can f II activityas described herein can be accomplished using one or more of severaldifferent assays. For example, in vitro, Can f I or Can f II T cellstimulatory activity is assayed by contacting a peptide known orsuspected of having a Can f I or Can f II activity with an antigenpresenting cell which presents appropriate MHC molecules in a T cellculture. Presentation of a peptide having a Can f I or Can f II activityin association with appropriate MHC molecules to T cells in conjunctionwith the necessary costimulation has the effect of transmitting a signalto the T cell that induces the production of increased levels ofcytokines, particularly of interleukin-2 and interleukin-4. The culturesupernatant can be obtained and assayed for interleukin-2 or other knowncytokines. For example, any one of several conventional assays forinterleukin-2 can be employed, such as the assay described in Proc.Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which areincorporated herein by reference. A kit for an assay for the productionof interferon is also available from Genzyme Corporation (Cambridge,Mass.).

Alternatively, a common assay for T cell proliferation entails measuringtritiated thymidine incorporation. The proliferation of T cells can bemeasured in vitro by determining the amount of ³H-labeled thymidineincorporated into the replicating DNA of cultured cells. Therefore, therate of DNA synthesis and, in turn, the rate of cell division can bequantified.

In one embodiment, peptides which have Can f I or Can f II T cellstimulating activity (i.e., the peptide comprises at least one T cellepitope) can be identified using an algorithm which predicts thepresence of T cell epitopes in a protein sequence, such as the algorithmdescribed by Hill et al., Journal of Immunology, 147:189-197 (1991). Thealgorithm of Hill et al. predicts the location of T cell epitopes in aprotein by the presence of certain patterns within the sequence whichare likely to bind MHC and therefore may contain T cell epitopes. Basedon the Hill et al. algorithm, two 13 amino acid peptides (discussed inExample 10) have been identified and produced synthetically. Suchpeptides were tested for T cell activity as described above (e.g., bymeasuring cellular uptake of tritiated thymidine). Specifically, inExample 10, human T cell stimulating activity was tested by culturing Tcells obtained from an individual sensitive to Can f I (i.e., anindividual who has an IgE mediated immune response to Can f I) with apeptide derived from Can f I and proliferation of T cells in response tothe peptide was determined, e.g., by measuring cellular uptake oftritiated thymidine. Stimulation indices for responses by T cells topeptides were calculated as the maximum CPM in response to a peptidedivided by the control CPM. A stimulation index (S.I.) equal to orgreater than two times the background level was considered “positive”.Positive results were used to calculate the mean stimulation index foreach peptide for the group of patients tested (See FIG. 25). Preferredpeptides of this invention comprise at least one T cell epitope and havea mean T cell stimulation index of greater than or equal to 2.0. Apeptide having a mean T cell stimulation index of greater than or equalto 2.0 in a significant number of dog dander allergen sensitive patientstested is considered useful as a therapeutic agent. Preferred peptideshave a mean T cell stimulation index of at least 2.5, more preferably atleast 3.0, more preferably at least 3.5, more preferably at least 4.0,more preferably at least 5.0 and most preferably at least about 6. Forexample, peptides having a Can f I activity and having a mean T cellstimulation index of at least 5, as indicated by data shown in FIG. 25,include Construct 1 (SEQ ID NO:105), Construct 2 (SEQ ID NO:106), andConstruct 3 (SEQ ID NO:107). T cell epitopes can also be predicted anddetermined as described above for peptides derived from Can f II.

In addition, preferred peptides have a positivity index (P.I.) of atleast about 60, more preferably about 100, more preferably at leastabout 200 and most preferably at least about 300. The positivity indexfor a peptide is determined by multiplying the mean T cell stimulationindex by the percent of individuals, in a population of individualssensitive to dog dander allergens (e.g., preferably a population of atleast 12 individuals, more preferably a population of at least 30individuals or more), who have a T cell stimulation index to suchpeptide of at least 2.0. Thus, the positivity index represents both thestrength of a T cell response to a peptide (S.I.) and the frequency of aT cell response to a peptide in a population of individuals sensitive todog dander allergens. In FIG. 10, the bar represents the positivityindex and the percent of individuals tested who have a T cellstimulation index of at least 2.0 to various peptides derived from Can fI. For example, as shown in FIG. 25, Peptide A0095 has a mean S.I. of3.0 and 43% of positive responses in the group of individuals testedresulting in a positivity index of 129.

In order to determine precise T cell epitopes by, for example, finemapping techniques, a peptide having Can f I or Can f II T cellstimulating activity and thus comprising at least one T cell epitope asdetermined by T cell biology techniques is modified by addition ordeletion of amino acid residues at either the amino or carboxy terminusof the peptide and tested to determine a change in T cell reactivity tothe modified peptide. Following this technique, peptides are selectedand produced recombinantly or synthetically. Peptides are selected basedon various factors, including the strength of the T cell response to thepeptide (e.g., stimulation index), the frequency of the T cell responseto the peptide in a population of individuals sensitive to dog danderallergens, and the potential cross-reactivity of the peptide with otherdog dander allergens. The physical and chemical properties of theseselected peptides (e.g., solubility, stability) are examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification as describedherein. The ability of the selected peptides or selected modifiedpeptides to stimulate human T cells (e.g., induce proliferation,lymphokine secretion) is then determined as described herein.

In another embodiment, a peptide having a Can f I or Can f II activityis screened for the ability to induce T cell non-responsiveness. Theability of a peptide known to stimulate T cells (as determined by one ormore of the above described assays), to inhibit or completely block theactivity of purified native Can f I or Can f II or portion thereof andinduce a state of non-responsiveness can be determined using subsequentattempts at stimulation of the T cells with antigen presenting cellsthat present native Can f I or Can f II or peptide having a Can f I orCan f II activity following exposure to the peptide, having a Can f I orCan f II activity. If the T cells are unresponsive to the subsequentactivation attempts, as determined by interleukin-2 synthesis and/or Tcell proliferation, a state of non-responsiveness has been induced. See,e.g., Gimmi et al., (1993) Proc. Natl. Acad. Sci USA, 90: 6586-6590; andSchwartz (1990) Science, 248: 1349-1356, for assay systems that can beused as the basis for an assay in accordance with the present invention.

In yet another embodiment, peptides having a Can f I or Can f IIactivity are identified by IgE binding activity. For therapeuticpurposes, peptides of the invention preferably do not bind IgE specificfor a dog dander allergen, or bind such IgE to a substantially lesserextent than the corresponding purified native dog dander allergen bindssuch IgE. Reduced IgE binding activity refers to IgE binding activitythat is less than that of purified native Can f I or Can f II protein.If a peptide having a Can f I or Can f II activity is to be used as adiagnostic reagent, it is not necessary that the peptide have reducedIgE binding activity compared to the native Can f I or Can f IIallergen. IgE binding activity of peptides can be determined by, forexample, an enzyme-linked immunosorbent assay (ELISA) using, forexample, sera obtained from a subject, (i.e., an allergic subject) thathas been previously exposed to the native Can f I or Can f II allergen.Briefly, the peptide suspected of having a Can f I or Can f II activityis coated onto wells of a microtiter plate. After washing and blockingthe wells, antibody solution consisting of the plasma of an allergicsubject who has been exposed to a peptide suspected of having a Can f Ior Can f II activity is incubated in the wells. The plasma is generallydepleted of IgG before incubation. A labeled secondary antibody is addedto the wells and incubated. The amount of IgE binding is then quantifiedand compared to the amount of IgE bound by a purified, native Can f I orCan f II protein. Alternatively, the IgE binding activity of a peptidecan be determined by Western blot analysis. For example, a peptidesuspected of having a Can f I or Can f II activity is run on apolyacrylamide gel using SDS-PAGE. The peptide is then transferred tonitrocellulose and subsequently incubated with sera from an allergicsubject. After incubation with a labeled secondary antibody, the amountof IgE bound is then determined and quantified.

Another assay which can be used to determine the IgE binding activity ofa peptide is a competition ELISA assay. Briefly, an IgE antibody pool isgenerated by combining plasma from dog dander allergic subjects thathave been shown by direct ELISA to have IgE reactive with native Can f Ior Can f II. This pool is used in ELISA competition assays to compareIgE binding of native Can f I or Can f II and a peptide suspected ofhaving a Can f I or Can f II activity. IgE binding for the native Can fI or Can f II protein and a peptide suspected of having a Can f I or Canf II activity is determined and quantified.

If a peptide having an activity of Can f I or Can f II binds IgE, and isto be used as a therapeutic agent, it is preferable that such bindingdoes not result in the release of mediators (e.g., histamines) from mastcells or basophils. To determine whether a peptide which binds IgEresults in the release of mediators, a histamine release assay can beperformed using standard reagents and protocols obtained, for example,from Amac, Inc. (Westbrook, Me.). Briefly, a buffered solution of apeptide suspected of having a Can f I or Can f II activity is combinedwith an equal volume of whole heparinized blood from an allergicsubject. After mixing and incubation, the cells are pelleted and thesupernatants are processed and analyzed using a radioimmunoassay todetermine the amount of histamine released.

Peptides having an activity of Can f I or Can f II which are to be usedas therapeutic agents are preferably tested in mammalian models of dogdander atopy, such as the mouse model disclosed in Tamura et al., (1986)Microbiol. Immunol., 30: 883-896, or in U.S. Pat. No. 4,939,239, or inthe primate model disclosed in Chiba et al., (1990) Int. Arch. AllergyImmunol., 93: 83-88. Initial screening for IgE binding to a peptidehaving an activity of Can f I or Can f II may be performed by scratchtests or intradermal skin tests on laboratory animals or humanvolunteers, or in in vitro systems such as RAST, RAST inhibition, ELISAassay, RIA (radioimmunoassay), or a histamine release assay, asdescribed above.

It is possible to modify the structure of a peptide having an activityof Can f I or Can f II for such purposes as increasing solubility,enhancing therapeutic or prophylactic efficacy, or stability (e.g.,shelf life ex vivo and resistance to proteolytic degradation in vivo).Such modified peptides are considered functional equivalents of peptideshaving an activity of Can f I or Can f II as defined herein. A modifiedpeptide can be produced in which the amino acid sequence has beenaltered, such as by amino acid substitution, deletion, or addition, tomodify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose.

For example, a peptide having an activity of Can f I or Can f II can bemodified so that it maintains the ability to induce T cellnon-responsiveness and bind MHC proteins without the ability to induce astrong proliferative response or possibly, any proliferative responsewhen administered in immunogenic form. In this instance, criticalbinding residues for T cell receptor function can be determined usingknown techniques (e.g., substitution of each residue and determinationof the presence or absence of T cell reactivity). Those residues shownto be essential to interact with the T cell receptor can be modified byreplacing the essential amino acid with another, preferably similaramino acid residue (a conservative substitution) whose presence is shownto enhance, diminish but not eliminate, or not affect T cell reactivity.In addition, those amino acid residues which are not essential for Tcell receptor interaction can be modified by being replaced by anotheramino acid whose incorporation may enhance, diminish but not eliminate,or not affect T cell reactivity, but does not eliminate binding torelevant MHC.

Additionally, a peptide having an activity of Can f I or Can f II can bemodified by replacing an amino acid shown to be essential to interactwith the MHC protein complex with another, preferably similar amino acidresidue (conservative substitution) whose presence is shown to enhance,diminish but not eliminate, or not affect T cell activity. In addition,amino acid residues which are not essential for interaction with the MHCprotein complex but which still bind the MHC protein complex can bemodified by being replaced by another amino acid whose incorporation mayenhance, not affect, or diminish but not eliminate T cell reactivity.Preferred amino acid substitutions for non-essential amino acidsinclude, but are not limited to substitutions with alanine, glutamicacid, or a methyl amino acid.

Another example of modification of a peptide having an activity of Can fI or Can f II is substitution of cysteine residues preferably withalanine, serine, threonine, leucine or glutamic acid residues tominimize dimerization via disulfide linkages. In addition, amino acidside chains of fragments of the protein of the invention can bechemically modified. Another modification is cyclization of the peptide.

In order to enhance stability and/or reactivity, a peptide having anactivity of Can f I or Can f II can be modified to incorporate one ormore polymorphisms in the amino acid sequence of the protein allergenresulting from any natural allelic variation. Additionally, D-aminoacids, non-natural amino acids, or non-amino acid analogs can besubstituted or added to produce a modified protein within the scope ofthis invention. Furthermore, a peptide having an activity of Can f I orCan f II can be modified using polyethylene glycol (PEG) according tothe method of A. Sehon and co-workers (Wie et al., supra) to produce aprotein conjugated with PEG. In addition, PEG can be added duringchemical synthesis of the protein. Other modifications of a peptidehaving an activity of Can f I or Can f II include reduction/alkylation(Tarr, Methods of Protein Microcharacterization, J. E. Silver ed.,Humana Press, Clifton N.J. 155-194 (1986)); acylation (Tarr, supra);chemical coupling to an appropriate carrier (Mishell and Shiigi, eds,Selected Methods in Cellular Immunology, W H Freeman, San Francisco,Calif. (1980), U.S. Pat. No. 4,939,239; or mild formalin treatment(Marsh, (1971) Int. Arch. of Allergy and Appl. Immunol., 41: 199-215).

To facilitate purification and potentially increase solubility of apeptide having an activity of Can f I or Can f II, it is possible to addan amino acid fusion moiety to the peptide backbone. For example,hexa-histidine can be added to the protein for purification byimmobilized metal ion affinity chromatography (Hochuli, E. et al.,(1988) Bio/Technology, 6: 1321-1325). In addition, to facilitateisolation of peptides free of irrelevant sequences, specificendoprotease cleavage sites can be introduced between the sequences ofthe fusion moiety and the peptide. In order to successfully desensitizea subject to Can f I or Can f II protein or related allergen, it may benecessary to increase the solubility of the protein by adding functionalgroups to the protein, or by omitting hydrophobic regions of theprotein.

To potentially aid proper antigen processing of T cell epitopes withinCan f I or Can f II, canonical protease sensitive sites can beengineered between regions, each comprising at least one T cell epitopevia recombinant or synthetic methods. For example, charged amino acidpairs, such as KK or RR, can be introduced between regions within aprotein or fragment during recombinant construction thereof. Theresulting peptide can be rendered sensitive to cleavage by cathepsinand/or other trypsin-like enzymes which would generate portions of theprotein containing one or more T cell epitopes. In addition, suchcharged amino acid residues can result in an increase in the solubilityof the peptide.

Site-directed mutagenesis of a nucleic acid encoding a peptide having anactivity of Can f I or Can f II can be used to modify the structure ofthe peptide by methods known in the art. Such methods may, among others,include polymerase chain reaction (PCR) with oligonucleotide primersbearing one or more mutations (Ho et al., (1989) Gene, 77: 51-59) ortotal synthesis of mutated genes (Hostomsky, Z. et al., (1989) Biochem.Biophys. Res. Comm, 161: 1056-1063). To enhance recombinant proteinexpression, the aforementioned methods can be applied to change thecodons present in the cDNA sequence of the invention to thosepreferentially utilized by the host cell in which the recombinantprotein is being expressed (Wada et al., supra).

Another aspect of the invention pertains to an antibody specificallyreactive with a peptide having an activity of Can f I or Can f II. Theantibodies of this invention can be used to standardize allergenextracts or to isolate the naturally-occurring or native form of Can f Ior Can f II. For example, by using peptides having an activity of Can fI or Can f II based on the cDNA sequence of Can f I or Can f II,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeusing standard methods. A mammal such as a mouse, a hamster or rabbitcan be immunized with an immunogenic form of the peptide (e.g., Can f Ior Can f II protein or an antigenic fragment which is capable ofeliciting an antibody response). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. A peptide having an activityof Can f I or Can f II can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassay can beused with the immunogen as antigen to assess the levels of antibodies.

Following immunization, anti-Can f I or anti-Can f II antisera can beobtained and, if desired, polyclonal anti-Can f I or anti-Can f IIantibodies isolated from the serum. To produce monoclonal antibodies,antibody producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, for example the hybridomatechnique originally developed by Kohler and Milstein, (1975) Nature,256: 495-497) as well as other techniques such as the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a peptide having anactivity of Can f I or Can f II and the monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with the peptide having anactivity of Can f I or Can f II. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having an anti-Canf I or anti-Can f II portion.

Another aspect of this invention provides T cell clones and soluble Tcell receptors specifically reactive with a peptide having an activityof Can f I or Can f II. Monoclonal T cell populations (i.e., T cellsgenetically identical to one another and expressing identical T cellreceptors) can be derived from an individual sensitive to Can f I or Canf II, followed by repetitive in vitro stimulation with a Can f I or Canf II protein or peptide having an activity of Can f I or Can f II in thepresence of MHC-matched antigen-presenting cells. Single Can f I or Canf II MHC responsive cells can then be cloned by limiting dilution andpermanent lines expanded and maintained by periodic in vitrorestimulation. Alternatively, Can f I or Can f II specific T-Thybridomas can be produced by a technique similar to B cell hybridomaproduction. For example, a mammal, such as a mouse, is immunized with apeptide having an activity of Can f I or Can f II, T cells are thenpurified and fused with an autonomously growing T cell tumor line. Fromthe resulting hybridomas, cells responding to a peptide having anactivity of Can f I or Can f II are selected and cloned. Procedures frompropagating monoclonal T cell populations are described in Cellular andMolecular Immunology (Abul K. Abbas et al. ed.), W. B. Saunders Company,Philadelphia, Pa. (1991) page 139. Soluble T cell receptors specificallyreactive with a peptide having an activity of Can f I or Can f II can beobtained by immunoprecipitation using an antibody against the T cellreceptor as described in Immunology: A Synthesis (Second Edition),Edward S. Golub et al., ed., Sinauer Associates, Inc., Sunderland, Mass.(1991) pages 366-269.

T cell clones specifically reactive with a peptide having an activity ofCan f I or Can f II can be used to isolate and molecularly clone thegene encoding the relevant T cell receptor. In addition, a soluble Tcell receptor specifically reactive with a peptide having an activity ofCan f I or Can f II can be used to interfere with or inhibitantigen-dependent activation of the relevant T cell subpopulation, forexample, by administration to an individual sensitive to Can f I or Canf II. Antibodies specifically reactive with such a T cell receptor canbe produced according to the techniques described herein. Suchantibodies can be used to block or interfere with the T cell interactionwith peptides presented by MHC.

Exposure of allergic subjects to peptides having an activity of Can f Ior Can f II and which have T cell stimulating activity, may cause theappropriate T cell subpopulations to become non-responsive to therespective protein allergen (e.g. fail to stimulate an immune responseupon such exposure). In addition, such administration may modify thelymphokine secretion profile as compared with exposure to thenaturally-occurring protein allergen or portion thereof (e.g., result ina decrease of IL-4 and/or an increase in IL-2). Furthermore, exposure topeptides having an activity of Can f I or Can f II which have T cellstimulating activity may influence T cell subpopulations which normallyparticipate in the response to the allergen such that these T cells aredrawn away from the site(s) of normal exposure to the allergen (e.g.,nasal mucosa, skin, and lung) towards the site(s) of therapeuticadministration of the protein or fragment derived therefrom. Thisredistribution of T cell subpopulations may ameliorate or reduce theability of an individual's immune system to stimulate the usual immuneresponse at the site of normal exposure to the allergen, resulting in adiminution in allergic symptoms.

A peptide having an activity of Can f I or Can f II when administered toa subject sensitive to dog dander allergens is capable of modifying theB cell response, T cell response, or both the B cell and the T cellresponse of the subject to the allergen. As used herein, modification ofthe allergic response of a subject to a dog dander allergen can bedefined as non-responsiveness or diminution in symptoms to the allergen,as determined by standard clinical procedures (See e.g., Varney et al.,(1990) British Medical Journal, 302: 265-269), including diminution indog dander induced asthmatic symptoms. As referred to herein, adiminution in symptoms includes any reduction in the allergic responseof a subject to the allergen following a treatment regimen with apeptide of the invention. This diminution in symptoms may be determinedsubjectively (e.g., the patient feels more comfortable upon exposure tothe allergen), or clinically, such as with a standard skin test.

Peptides or antibodies of the present invention can also be used fordetecting and diagnosing sensitivity to Can f I or Can f II. Forexample, this could be done by combining blood or blood productsobtained from a subject to be assessed for sensitivity with peptidehaving an activity of Can f I or Can f II, under conditions appropriatefor binding of components in the blood (e.g., antibodies, T cells, Bcells) with the peptide(s) and determining the extent to which suchbinding occurs. Other diagnostic methods for allergic diseases which thepeptides or antibodies of the present invention can be used includeradio-allergosorbent test (RAST), paper radioimmunosorbent test (PRIST),enzyme linked immunosorbent assay (ELISA), radioimmunoassays (RIA),immuno-radiometric assays (IRMA), luminescence immunoassays (LIA),histamine release assays and IgE immunoblots.

The present invention further provides methods of detecting and treatingsensitivity in a subject to Can f I or Can f II. The presence insubjects of IgE specific for Can f I or Can f II and the ability of Tcells of the subjects to respond to T cell epitopes of Can f I or Can fII can be determined by administering to the subject an Immediate TypeHypersensitivity test and/or a Delayed Type Hypersensitivity test (Seee.g., Immunology (1985) Roitt, I. M., Brostoff, J., Male, D. K. (eds),C. V. Mosby Co., Gower Medical Publishing, London, N.Y., pp. 19.2-19.18;pp. 22.1-22.10) utilizing a peptide having an activity of Can f I or Canf II, or a modified form of a peptide having an activity of Can f I orCan f II, each of which binds IgE specific for the allergen. The samesubjects are administered a Delayed Type Hypersensitivity test prior to,simultaneously with, or subsequent to administration of the ImmediateType Hypersensitivity test. Of course, if the Immediate TypeHypersensitivity test is administered prior to the Delayed TypeHypersensitivity test, the Delayed Type Hypersensitivity test would begiven to those subjects exhibiting a specific Immediate TypeHypersensitivity reaction. The Delayed Type Hypersensitivity testutilizes a peptide having an activity of Can f I or Can f II which hashuman T cell stimulating activity and which does not bind IgE specificfor the allergen in a substantial percentage of the population ofsubjects sensitive to the allergen (e.g., at least about 75%). Thosesubjects found to have both a specific Immediate type Hypersensitivityreaction and a specific Delayed Type Hypersensitivity reaction areadministered an amount of a composition suitable for pharmaceuticaladministration. The composition comprises the peptide having an activityof Can f I or Can f II as used in the Delayed Type Hypersensitivity testand a pharmaceutically acceptable carrier or diluent.

A peptide having an activity of Can f I or Can f II can be used inmethods of diagnosing, treating, and preventing allergic reactions to adog dander allergen or a cross-reactive protein allergen. Thus, thepresent invention provides compositions suitable for pharmaceuticaladministration comprising an amount of at least one peptide having anactivity of Can f I or Can f II and a pharmaceutically acceptablecarrier. Administration of the compositions of the present invention toa subject to be desensitized can be carried out using known procedures,at dosages and for periods of time effective to reduce sensitivity(i.e., reduce the allergic response) of the subject to a dog danderallergen. The term subject is intended to include living organisms inwhich an immune response can be elicited, e.g., mammals. Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. An amount of at least one peptide having an activity of Can f Ior Can f II necessary to achieve a therapeutic effect may vary accordingto factors such as the degree of sensitivity of the subject to dogdander, the age, sex, and weight of the subject, and the ability of apeptide having an activity of Can f I or Can f II to elicit an antigenicresponse in the subject. Dosage regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound (i.e., a peptide having an activity of Can f I orCan f II) may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active compound may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound.

To administer a peptide having an activity of Can f I or Can f II byother than parenteral administration, it may be necessary to coat thepeptide with, or co-administer the peptide with, a material to preventits inactivation. For example, a peptide having an activity of Can f Ior Can f II may be administered to an individual in an appropriatecarrier, diluent or adjuvant, co-administered with enzyme inhibitors orin an appropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes (Strejan et al., (1984) J. Neuroimmunol., 7: 27).For purposes of inducing T cell nonresponsiveness, the composition ispreferably administered in non-immunogenic form, e.g., one that does notcontain adjuvant.

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (i.e., a peptide having an activity of Can f I or Can f II) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient (i.e., atleast one peptide having an activity of Can f I or Can f II) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

When the peptide having an activity of Can f I or Can f II is suitablyprotected, as described above, the peptide may be orally administered,for example, with an inert diluent or an assimilable edible carrier. Thepeptide and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the individual's diet. For oral therapeutic administration, theactive compound may be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 5 to about 80% of the weight of the unit.The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in subjects.

The present invention also provides a composition comprising at leasttwo peptides having an activity of Can f I or Can f II (e.g., a physicalmixture of at least two peptides), each having T cell stimulatingactivity. For example, at least two peptides each having as activity ofCan f I can be combined or at least two peptides each having an activityof Can f II can be combined, or at least one peptide having an activityof Can f I and at least one peptide having an activity of Can f II canbe combined and administered. Alternatively, a peptide having at leasttwo regions, each having T cell stimulating activity (i.e., each regioncomprising at least one T cell epitope) can be administered to anallergic subject. Such a peptide can have at least two regions derivedfrom the same allergen, Can f I or Can f II, or a combination of Can f Iand Can f II. A composition of two peptides or a peptide having at leasttwo regions can be administered to a subject in the form of acomposition with a pharmaceutically acceptable carrier as hereinbeforedescribed. An amount of one or more of such compositions can beadministered simultaneously or sequentially to a subject sensitive to adog dander allergen to treat such sensitivity.

The cDNA (or the mRNA which served as a template during reversetranscription) encoding a peptide having an activity of Can f I or Can fII can be used to identify similar nucleic acid sequences in any varietyor type of animal and, thus, to molecularly clone genes which havesufficient sequence homology to hybridize to the cDNA encoding a peptidehaving an activity of Can f I or Can f II. Thus, the present inventionincludes not only peptides having an activity of Can f I or Can f II,but also other proteins which may be allergens encoded by DNA whichhybridizes to DNA of the present invention.

Isolated peptides that are immunologically related to Can f I or Can fII, such as by antibody cross-reactivity or T cell cross-reactivity,other than those already identified, are within the scope of theinvention. Such peptides bind antibodies specific for the protein andpeptides of the invention, or stimulate T cells specific for the proteinand peptides of this invention.

A peptide having an activity of Can f I or Can f II (i.e., Can f I orCan f II produced recombinantly or by chemical synthesis) is free of allother dog dander proteins and, thus, is useful in the standardization ofallergen extracts which are key reagents for the diagnosis and treatmentof dog dander hypersensitivity. In addition, such a peptide is of aconsistent, well-defined composition and biological activity for use inpreparations which can be administered for therapeutic purposes (e.g.,to modify the allergic response of a subject sensitive to dog dander).Such peptides can also be used to study the mechanism of immunotherapyof Canis familiaris allergy and to design modified derivatives oranalogs useful in immunotherapy.

Work by others has shown that high doses of allergen extracts generallyproduce the best results during immunotherapy (i.e., best symptomrelief). However, many subjects are unable to tolerate large doses ofsuch extracts due to systemic reactions elicited by the allergens andother components within these preparations. A peptide having an activityof Can f I or Can f II of the invention has the advantage of being freeof all other dander protein. Thus, such a peptide can be administeredfor therapeutic purposes.

It is now also possible to design an agent or a drug capable of blockingor inhibiting the ability of a dog dander allergen to induce an allergicreaction in sensitive subjects. Such agents could be designed, forexample, in such a manner that they would bind to relevant anti-Can f Ior anti-Can f II IgE molecules, thus preventing IgE-allergen binding,and subsequent mast cell/basophil degranulation. Alternatively, suchagents could bind to cellular components of the immune system, resultingin suppression or desensitization of the allergic responses to dogdander allergens. A non-restrictive example of this is the use ofpeptides including B or T cell epitopes of Can f I or Can f II, ormodifications thereof, based on the cDNA protein structure of Can f I orCan f II to suppress the allergic response to a dog dander allergen.This could be carried out by defining the structures of fragmentsencoding B and T cell epitopes which affect B and T cell function in invitro studies with blood components from subjects sensitive to dogdander.

The invention is further illustrated by the following examples whichshould not be construed as further limiting the subject invention. Thecontents of all references and published patent applications citedthroughout this application are hereby incorporated by reference.

EXAMPLE 1 Protein Sequence Analysis of Purified Can f I

Affinity purified Can f I protein was obtained from Dr. Aalberse (deGroot, H. et al., supra). An Applied Biosystems Model 477A gas phasesequencer with on-line phynylthiohydantoin (HTH) amino acid analysis(Model 120A) was used to sequence the purified Can f I protein. Amodification of the extraction program, multiple butylchlorideextractions, was used to improve the amino acid recovery.O-phthaladehyde (OPA) was used in blocking of primary amines whenproline was located at the amino terminus. Brauer, A. W., et al., (1984)Anal. Biochemistry, 137: 134, 142. In situ alkylation was performed byusing the non-nucleophilic reductant, tributylphosphine with concomitantalkylation by 4-vinyl pyridine in ethylmorpholine buffer. Andrews, P. C.and Dixon, J. E., (1987) Anal. Biochemistry, 161: 524-528.

Using this methodology, the sequence of the N-terminus of the Can f Iprotein was determined, contrary to previous reports that the N-terminusis blocked (Schou, C. et al., supra). The N-terminal sequence of 65amino acid residues which was identified through multiple N-terminalsequence analysis in conjunction with OPA blocking of contaminatingsignal represents a novel protein sequence (FIG. 5). The Can f I proteinsequence was confirmed and expanded by sequence analysis of CNBr cleavedpeptides. In situ CNBr digestion of Can f I on the sequence glass filterdisk provided additional protein sequence information. Simpson, R. J.and Nice, E. C., (1984) Biochem. International, 8: 787-791. Prior to thein situ CNBr cleavage, forty-four cycles of amino acid sequencing wereperformed and then the protein sample was treated with OPA to block allamino groups. After five hours of in situ CNBr digestion, three majorsequences were identified corresponding to the sequences after Met46,Met30 and an unknown Met, later shown to be Met103. An additional OPAblock after cycle 18 (before Pro65) extended the sequence to Asp86.Sequence analysis of CNBr peptide fragments isolated by HPLC (AppliedBiosystem, Inc., Model 130, C8 Column) further extended the N-terminalsequence to ninety-four amino acid residues. In situ CNBr cleavage inconjunction with OPA blocking also identified a 39 amino acid residuepeptide (residues 104-142). A potential N-glycosylation site was foundin the cDNA deduced amino acid sequence, Asn54-Ile55-Thr56. The proteinsequence analysis identified the Ile55 and Thr56 of Can f I, however,nothing could be identified at the position 54. This suggests thatpost-translation modification occurs at Asn54 of Can f I and themodification is stable to the trifluoroacetic acid treatment duringprotein sequencing.

EXAMPLE 2 Extraction of mRNA from Canine Parotid Glands and Cloning ofCan f I

A pair of fresh parotid glands from a single outbred dog were obtainedfrom the Tufts University School of Veterinary Medicine (Worcester,Mass.) and, washed in phosphate buffered saline, and immediately frozenon dry ice. RNA was extracted essentially as described in the literature(Chirgwin, J. M. et al., (1979) Biochemistry, 18: 5294-5299.). One glandwas pulverized to a powder with a mortar and pestle frozen in liquid N2,and suspended in 25 ml of GTC buffer (50% w/v guanidine thiocynate, 0.5%w/v Na lauryl sarcosine, 0.7% v/v β-mercaptoethanol, 0.1% v/v SigmaAntifoam A, 25 mM Na citrate, pH 7.0) and vortexed until dissolved.Genomic DNA present in the solution was sheared by forcing the solutionthrough a 16 gauge needle until the viscosity of the solution no longerdecreased. The sheared solution was centrifuged at 3 K rpm for 5 minutesat room temperature. The supernatant was then sheared further through a23 gauge needle until its viscosity no longer decreased, and cleared bycentrifugation at 5 K rpm for 5 minutes at room temperature. Thesolution was layered onto a CsCl cushion (5.7 M CsCl, 10 mM EDTA pH 7.5)and ultracentrifuged in a Beckman SW 41 Ti rotor at 35 K rpm for 16hours at 20° C. The supernatant was discarded and the RNA pellet washedin 70% EtOH then resuspended in 0.3 M NaOAc, 10 mM EDTA, 0.1% SDS. Twovolumes of absolute EtOH were added, and precipitation carried out ondry ice. RNA was pelleted by centrifugation, 70% EtOH washed, andresuspended in TES (10 mM Tris, 1 mM EDTA, 0.1% SDS). The final yieldwas ˜1.8 mg.

Single strand total dog parotid gland cDNA was synthesized using theabove RNA preparation as a template in reverse transcription. 4 μg oftotal RNA were EtOH precipitated (using glycogen as carrier: molecularbiology grade Boerhinger Manheim), 70% EtOH washed, and resuspended in10 μl of dH2O. Oligo dT(12-18) was added to 50 μg/ml and the RNAdenatured at 70° C. for 5 minutes. The reaction was quick chilled on iceand 1 μl (40 units) of RNAsin (Promega) was added as a prophylacticagainst contaminating RNases. The components from the BRL Superscript™Reverse Transcriptase Kit were added as follows: 4 μl 5×buffer, 2 μl 0.1M DTT, 1 μl 10 mM dNTP mix. After warming the reaction to 37° C., 1 μl(200 units) of Superscript™ Reverse Transcriptase was added, and thereaction allowed to proceed for one hour at 37° C. Reverse transcriptionwas terminated by incubation at 70° C. for 15 minutes, and the reactionstored at −20° C.

Initially the MOPAC (mixed oligonucleotides primed amplification ofcDNA) technique of PCR amplification (Lee, C. C. et al., (1988) Science,239: 1288-1291) was used to obtain a partial cDNA of Can f I encodingamino acids 14 to 29 of the mature protein. Using dog parotid cDNA as atemplate with degenerate primer pairs (synthesized on an AppliedBiosystems 392) based on residues 9 to 15 (SEQ ID NO:3) [FIG. 1, S1A(SEQ ID NO:4) or S1B (SEQ ID NO:5)] and 30 to 37 (SEQ ID NO:10) [FIG. 1,AS2A (SEQ ID NO:11) or AS2B (SEQ ID NO:12)] of mature Can f I, a DNAfragment of the predicted size (˜3×28 amino acids, or 84 bp) could beamplified using a PCR kit (GeneAmp kit, Perkin Elmer Cetus, Norwalk,Conn.) in conjunction with the following program in an MJ ResearchMinicycler: 40×(92° C. 30 seconds/55° C. 1 minutes/75° C. 1 minutes).The primer pair 5′ S1B/3′ AS2B amplified the predicted fragment with thegreatest efficiency, inferring that in both coding regions, the leucineresidue was encoded by CTX rather than TT (A or G). As a test of itsauthenticity, the amplified fragment hybridized on a Southern blot to aninternal degenerate oligonucleotide probe Dog probe 1 (SEQ ID NO:7),[based on Can f I residues 17 to 24, (SEQ ID NO:6)] that had been endlabeled with γ-32P ATP using T4 polynucleotide kinase. After subcloningof the amplified fragment into Bluescript KS plasmid vector (Stratagene,San Diego, Calif.), it was sequenced using a Sanger dideoxy terminationkit (USB Cleveland, Ohio) and shown to correctly encode residues 16 to29 of mature Can f I.

Similarly, when amino acid sequence analysis of purified Can f I yieldedsequence information extending to residue 94 of the mature protein, newprimer pairs were used in MOPAC PCR amplification of an extended partialCan f I cDNA (residues 14 to 87). The 5′ or sense primers SA (residues14 to 20) and SB (residues 21 to 27) were a nested pair based on theknown Can f I partial cDNA sequence, while the 3′ or antisense primers,AS3A (SEQ ID NO:13) (FIG. 1) and AS3B (SEQ ID NO:14) (FIG. 1) weredegenerate oligonucleotides based on residues 88 to 94. In sequentialrounds of PCR ( 1/100th of the first reaction was used as template forthe second reaction) using conditions described above in a pair ofsuccessive reactions using nested 5′ sense oligos in conjunction with asingle 3′ antisense degenerate primer, a DNA fragment of the predictedsize (˜3×80 amino acids, or 240 bp) could be amplified. Degenerate 3′antisense oligo AS3B was more efficient in collaborating with thesuccessive pairs of 5′ sense oligos to amplify the partial internal Canf I cDNA than oligo AS3A, again suggesting that the leucine residue wasencoded by CTX rather than TT(A or G). The 240 bp DNA fragment wassubcloned into Bluescript KS plasmid vector and sequenced as describedabove. It too proved to be an authentic Can f I cDNA. The missingresidue in the amino acid sequence of Can f I at residue 54 wasdetermined to be an asparagine on the grounds that: 1) no amino acidsignal was found at residue 54 during protein sequence analysis; and 2)the asparagine residue resides within a consensus sequence for N-linkedglycosylation (N54 I55 T56). These data strongly suggest that the N54residue is modified by N-linked glycosylation.

To obtain the 3′ portion of the Can f I cDNA, the RACE (RapidAmplification of cDNA ends) PCR protocol was employed (Frohman, M. A. etal., (1988) Proc. Natl. Acad. Sci., 85: 8998-9002). First strand cDNAsynthesis from total dog parotid RNA was carried out as described above,except that the JM3 oligonucleotide was substituted for oligo dT as theprimer in the reaction. The JM3 primer (SEQ ID NO:22) has an arbitrarytract of ˜40 nucleotides encoded 5′ of an oligo dT tract (FIG. 2).Hence, upon priming of poly A+RNA to make cDNA, this known nucleotidetag is covalently linked to the 5′ end of the nascent cDNA transcripts.Using nested 5′ primers, SD (residues 73-79 (SEQ ID NO:15)) and SE(residues 80-86 (SEQ ID NO:17)), based on known Can f I cDNA sequencefrom MOPAC PCR analysis and nested primers based on the known JM3 primersequence (JM3-1 (SEQ ID NO:23) and JM3-3 Bam (SEQ ID NO:24)) in PCRamplification as above (except the PCR program was 40×[92° C. 30seconds/60° C. 1 minutes/75° C. 1 minutes]), a DNA fragment ˜500 bp inlength was amplified. When probed against a kinase labeled degenerateoligonucleotide, Dog Probe 2 (SEQ ID NO:9) [residues 88 to 94 of matureCan f I (SEQ ID NO:8)], this band proved positive for hybridization.Upon subcloning into plasmid vector and DNA sequence analysis, threedifferent partial 3′ Can f I cDNAs were identified: Can f I (SEQ IDNO:61), 2Can f I (SEQ ID NO:62), and 3Can f I (SEQ ID NO:63), each asshown in FIG. 9. 2Can f I had a sequence that encoded a methionineresidue followed by an asparagine-proline pair. These landmark residuesfor protein sequence analysis predicted: 1) a CNBr fragment with the NH2terminal sequence MAKLLGRDPEQ . . . (SEQ ID NO:64); 2) an acid sensitivecleavage site at the DP pair; and 3) a proline residue which shouldprove refractory to OPA treatment and yield amino acid sequence datawhere all other NH2 termini would be blocked by the treatment. Indeed,further protein sequence analysis of purified Can f I did identify aCNBr fragment that in conjunction with OPA blockage at the internalproline residue had the sequence (M)AKLLGRDPEQSQEALEDF( )EFS()AKGLNQEILELAQS(E)T (SEQ ID NO:65). Acid cleavage of the purifiedprotein yielded a peptide with the sequence (D)PEQS(E)EA (SEQ ID NO:66).These complimentary data from protein sequence analysis and partial cDNAcloning of Can f I indicated that the authentic 3′ end of the Can f IcDNA may not have been isolated.

Comparison of the amino acid sequence data from sequencing purified Canf I and those encoded by the partial cDNAs 2Can f I and 3Can f Iinferred the origin of the multiple species of 3′ cDNAs may have beenalternative splicing of the nascent Can f I transcript (note how inpartial cDNA 3Can f I residues from the NH2 terminus of the CNBrfragment are found linked to the fragment's COOH terminal residueswithout the intervening residues). In contrast to this hypothesis ofmultiple cDNAs originating at the level of alternative splicing, theabove PCR amplification of the 3′ end of the Can f I cDNA produced asingle prominent DNA fragment ˜500 bp in length. However, the threepartial 3′ cDNAs were either significantly longer or shorter than 500bp. This suggested rare partial cDNAs were being subcloned, perhapsbecause the authentic Can f I cDNA harbored the restrictions site(s)encoded at the ends of the primers used in subcloning of DNA fragmentsthat arise from PCR amplification. Hence, when digesting the PCR productrepresenting the authentic Can f I cDNA with restriction endonucleases(in this case 5′ EcoR I and 3′ BamH I) one would 1) cut the authenticCan f I cDNA into at least two pieces, and 2) bias towards subcloningrare cDNAs that had arisen from alternative splicing of the nascent Canf I RNA transcripts that had exons containing EcoR I and/or BamH I sitesdeleted. To address this situation, new primers with differentrestriction enzyme sites at their 5′ ends were synthesized and used inRACE PCR of the 3′ end of the Can f I cDNA. The JM3-3 oligo wasresynthesized with a Bgl II linked to its 5′ end [JM3-3XB (SEQ IDNO:21)] (FIG. 2), while the 5′ primers SD and SE were resynthesized withXho I sites at their 5′ ends [XSD (SEQ ID NO:16) and XSE (SEQ ID NO:18)](FIG. 2). After nested PCR of JM3 primed total dog parotid cDNA usingthese new primers and the previous amplification conditions, the intact3′ end of the Can f I cDNA (which hybridized to kinase labeled Dog Probe4, [(SEQ ID NO:20), residues 115-121 of mature Can f I (SEQ ID NO:19)(FIG. 2)] was subcloned and sequenced. The translated amino acidsequence of the partial cDNA corresponded directly with the proteinsequence data and extended it a further 6 amino acids beforeencountering a stop codon. As the cloning artifacts had predicted, bothEcoR I and BamH I sites were found in the coding region of the intact 3′Can f I cDNA.

The 5′ end of the Can f I cDNA was cloned using an anchored PCRtechnique (Roux, K. H. and Dhanarajan, P., (1990) Biotechniques, 8:48-57; Rafnar, T. et al., (1991) J. Biol. Chem., 266: 1229-1236). Doublestrand dog parotid cDNA was synthesized using a kit (BRL SuperscriptcDNA Synthesis System) employing the method of RNase H priming of thesecond strand of cDNA synthesis (Gubler, U., and Hoffman, B. J., (1983)Gene, 25: 263-269). The blunt double stranded cDNA was ligated to ananchor adapter, thereby placing a known sequence at the 5′ ends of cDNAs(SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27) (see FIG. 3). A primerbased on the anchor sequence was used as a 5′ sense primer (AP) inconjunction with a nested pair of 3′ antisense primers, ASA (SEQ IDNO:31) [residues 18 to 24 (SEQ ID NO:30)] and ASB (SEQ ID NO:33)[residues 25 to 30 (SEQ ID NO:32)] based on known Can f I cDNA sequencefrom MOPAC PCR in sequential rounds of PCR (40×[92° C. 30 seconds/60° C.1 minutes/75° C. 1 minutes]) to amplify the 5′ end of the Can f I cDNA(1° reaction ds anchored cDNA template with 5′ AP/3′ ASB primers: 2°reaction 1/100th 1° reaction template with 5′ AP/3′ ASA primers).Agarose gel electrophoresis analysis of the 2° reaction revealed a broadband ˜300 bp in length, which in Southern blot analysis hybridized to a32P kinased degenerate oligonucleotide probe, Dog Probe 0 (SEQ ID NO:29)(FIG. 3), based on residues 9 to 17 (SEQ ID NO:28) of mature Can f I.The amplified fragment was subcloned into Bluescript KS plasmid andsubjected to DNA sequence analysis. It's authenticity as the 5′ end ofthe Can f I cDNA was confirmed by the presence of the first 13 residuesof mature Can f I protein at the 3′ end of the partial cDNA. Sequence ofthe longest partial 5′ cDNA extended a further 126 bp and encoded a 26amino acid leader sequence not found in mature Can f I. Although noin-frame stop codons were found 5′ of the presumed initiator methioninecodon (M-26), it is presumed to be the true initiator codon and not justan internal methionine residue because: 1) it is embedded within aconsensus sequence for translation initiation in mammalian cells (Kozak,M., (1986) Cell, 44: 283-292); and 2) the predicted leader sequence ishighly homologous to the leader sequences of proteins that are highlyrelated to Can f I (see below).

A contiguous Can f I cDNA was then amplified and both strands directlysequenced as a PCR product to confirm the coding sequence of themolecule. To minimize the possibility of introducing errors in theamplified cDNA during the PCR reaction, Pfu I DNA polymerase(Stratagene, San Diego, Calif.) was used to amplify the coding cDNA. PfuI DNA polymerase has been documented to introduce an order of magnitudefewer errors than Taq DNA polymerase during PCR (Lundberg, K. S., (1991)Gene, 108: 1-4). Direct sequencing of non-cloned DNA fragments from PCRreactions should also obviate any errors made by DNA polymerases duringPCR since such errors will be scattered at random throughout thepopulation of PCR products (Gyllensten, U. B., and Ehrlich, H. A.,(1988) Proc. Natl. Acad. Sci. USA, 85: 7652-7656). Primers used in theamplification/sequencing included the 5′ sense leader ex oligo (SEQ IDNO:35) [residues −26 to −20 of Can f I (SEQ ID NO:34)] and the 3′antisense stop Bgl II oligo (SEQ ID NO:36) [a 24-mer 40 bp 3′ of thestop codon of Can f I] (FIG. 4). A program of 40×(95° C. 30 seconds/60°C. 45 seconds/75° C. 45 seconds) was used with the aforementionedprimers and Pfu I DNA polymerase to amplify a DNA fragment ˜600 bp inlength, which was subsequently isolated as a band on a 0.6% low meltagarose gel. This gel slice was melted at 70° C. and used as templatefor PCR sequencing using 32P labelled oligonucleotides as primers and acommercially available kit (AmpliTaq Cycle Sequencing Kit, Perkin ElmerCetus, Norwalk, Conn.). A program of 30×(95° C. 30 seconds/60° or 68° C.30 seconds) was used for the cycle sequencing. The PCR sequencingstrategy to obtain unambiguous sequence of the mature Can f I proteinfrom both strands of the amplified cDNA is depicted in FIG. 4 with thefollowing sense primers: start ex (SEQ ID NO:37); SB (SEQ ID NO:38); SK(SEQ ID NO:39); SE (SEQ ID NO:40); and SH (SEQ ID NO:41), and thefollowing antisense primers: Dog 9 (SEQ ID NO:42); ASK (SEQ ID NO:43);ASB (SEQ ID NO:44); and ASJ (SEQ ID NO:45). PCR cycle sequencinganalysis of amplified cDNA encoding the mature Can f I protein served toconfirm the DNA sequence obtained previously from cloned partial cDNAsof Can f I, FIG. 4.

In order to infer the possible biological function of Can f I, its aminoacid sequence was compared to those in the GenBank, GenBankUpdate, EMBL,and EMBL Update sequence data bases (as of Jun. 25, 1992) using the NCBIBLAST network service (Altschul, S. F., et al., (1990) J. Mol Biol.,215: 403-410). Can f I precursor protein (including the signal sequencenot found in mature Can f I protein) displayed strong homology to threeproteins: 1) Human von Ebner's gland protein; 2) Rat (VEG) von Ebner'sgland protein precursor (Hartwig, S., et al., (1990) Nature, 343:366-369); and 3) Rat odorant-binding protein (Pevsner, J., et al.,(1988) Science, 241: 336-339). von Ebner's gland is a sublingual glandand secretes an abundant protein into the saliva speculated to beinvolved in potentiating the sense of taste involving hydrophobicmolecules. von Ebner's gland protein belongs to a superfamily of oflipophilic molecule carriers (Godovac-Zimmermann, J., (1988) TrendsBiochem. Sci., 13: 64-66). The homology between Can f I and the humanand rat von Ebner's gland proteins indicates that Can f I may be thecanine homolog of von Ebner's gland protein. Additional data indicatesthat Can f I mRNA is expressed predominantly in the tongue epithelialtissue where von Ebner's glands are localized and only at a very lowlevel (not detectable by Northern blot analysis) in parotid glands.

EXAMPLE 3 Protein Sequence Analysis of Purified Can f II

Affinity purified Can f II protein was obtained from Dr. Aalberse (deGroot, H. et al., supra). An Applied Biosystems Model 477A gas phasesequencer with on-line phynylthiohydantoin (HTH) amino acid analysis(Model 120A) was used to sequence the purified Can f II protein. Amodification of the extraction program, multiple butylchlorideextractions, was used to improve the amino acid recovery.O-phthaladehyde (OPA) was used in blocking of primary amines whenproline was located at the amino terminus. Brauer, A. W., et al., (1984)Anal. Biochemistry, 137: 134, 142. In situ alkylation was performed byusing the non-nucleophilic reductant, tributylphosphine with concomitantalkylation by 4-vinyl pyridine in ethylmorpholine buffer. Andrews, P. C.and Dixon, J. E., (1987) Anal. Biochemistry, 161: 524-528.

Using this methodology, the sequence of the N-terminus of the Can f IIprotein was determined. The N-terminal sequence of 38 amino acidresidues which was identified through multiple N-terminal sequenceanalysis in conjunction with OPA blocking of contaminating signalrepresents a novel protein sequence FIG. 19 (SEQ ID NO: 88).

EXAMPLE 4 Extraction of mRNA from Canine Parotid Glands and Cloning ofCan f II

The strategy used to clone Can f II is schematically drawn in FIG. 14.cDNA was synthesized using the above preparation as a template inreverse transcription. In the next step ds cDNA was used as a templatefor PCR along with degenerate primers which were designed based on aminoacid sequence of Can f II and oriented to amplify a fragment of Can f IIcDNA. PCR product was gel purified and than subjected to directsequencing. The nucleotide sequence confirmed that the PCR productrepresents a fragment of Can f II cDNA. Further polymerase chainreactions were performed using Can f II specific primers in order toobtain a longer fragment which was subsequently used as a probe toscreen a dog cDNA library. Positive clones were identified, plaquepurified, sequenced and full length Can f II cDNA was obtained.

Fresh parotid glands from a single outbred dog were obtained from theTufts University School of Veterinary Medicine (Worcester, Mass.),washed in phosphate buffered saline, and immediately frozen on dry ice.RNA was extracted essentially as described in the literature (Chirgwin,J. M. et al., (1979) Biochemistry, 18: 5294-5299.). Two glands (approx.50 g) were pulverized to a powder with a mortar and pestle frozen inliquid N2, and suspended in 25 ml of GTC buffer (50% w/v guanidinethiocynate, 0.5% w/v Na lauryl sarcosine, 0.7% v/v b-mercaptoethanol,0.1% v/v Sigma Antifoam A (Sigma, St. Louis Mo.), 25 mM Na citrate, pH7.0) and vortexed until dissolved. Genomic DNA present in the solutionwas sheared by forcing the solution through a 16 gauge needle until theviscosity of the solution no longer decreased. The sheared solution wascentrifuged at 3 K rpm for 5 minutes at room temperature. Thesupernatant was then sheared further through a 23 gauge needle until itsviscosity no longer decreased, and cleared by centrifugation at 5 K rpmfor 5 minutes at room temperature. The solution was layered onto a CsClcushion (5.7 M CsCl, 10 mM EDTA pH 7.5) and centrifuged in a Beckman SW41 Ti rotor at 35 K rpm for 16 hours at 17° C. The supernatant wasdiscarded and the RNA pellet washed in 70% EtOH then resuspended in 0.3M NaOAc, 10 mM EDTA, 0.1% SDS. Two volumes of absolute EtOH were added,and precipitation carried out on dry ice. RNA was pelleted bycentrifugation, 70% EtOH washed, and resuspended in TES (10 mM Tris, 1mM EDTA, 0.1% SDS). The final yield was ˜5.3 mg of total RNA. mRNA wasisolated from total RNA by chromatography on oligo(dT) cellulose usingthe method described by Aviv, H. and Leder, P. (Proc. Natl. Acad. Sci.USA, (1972) 69: 1408). 20 μg of poly (A) RNA was obtained from 1.7 mg oftotal RNA.

The conversion of gland poly(A) mRNA into double stranded cDNA wascarried out using standard procedure (Ausubel et al., (1993) CurrentProtocols in Molecular Biology, John Wiley & Sons). First, poly(A) RNAwas copied onto cDNA was using Amersham cDNA Synthesis System Plusaccording to the manufacturer's procedure. 4 μg of poly (A) RNA was usedas a template and oligo dT(12-18) was used to prime first strandsynthesis. The RNA in RNA/DNA hybrid was than removed by RNaseH and thesecond strand was synthesized by DNA Polymerase I. Double stranded cDNAwas completed and made blunt by T4 DNA polymerase and E. coli DNA ligaseaccording to Gubler, U. and Hoffman, B. J., (1983) Gene, 25: 263).

Initially, PCR amplification (Mullis, K. B. and Faloona, F., (1987)Methods Enzymol, 155: 355-360) was used to obtain a partial cDNA of Canf II encoding amino acids 16 to 29 (SEQ ID NO: 97) of the matureprotein. Using dog parotid cDNA as a template with degenerate primerpairs (synthesized on an Applied Biosystems 392) based on residues 3 to6 (S1A) (FIG. 13) (SEQ ID NO: 91) and (S1B) (FIG. 13) (SEQ ID NO: 92)and on residues 33 to 38 (ASP2A) (FIG. 13) (SEQ. ID NO: 93) and (ASP2B)(FIG. 13) (SEQ ID NO: 94) of mature Can f II, a DNA fragment of thepredicted size (˜120 bp) was amplified using a PCR kit (GeneAmp kit,Perkin Elmer Cetus, Norwalk, Conn.). Conditions for the reaction were:denaturation for 1 minute at 94° C.; annealing for 1 minute at 42° C.and polymerization for 1 minute at 72° C. The cycle was repeated 30times. As a test of its authenticity, the amplified fragment wassubjected to direct sequencing using a commercially available kit(AmpliTaq Cycle Sequencing Kit, Perkin Elmer Cetus, Norwalk, Conn.)according to the instructions supplied. Primers used in theamplification/sequencing (which included S1A, S1B, ASP2A and ASP2B) hadbeen end labeled with γ-32P ATP using T4 polynucleotide kinase. Thefollowing program of 19 cycles (denaturation at 95° C. for 1 minute;annealing at 50° C. for 1 minute and extension at 72° C. for 15 seconds)was used for the cycle sequencing in a MJ Research Minicycler. Thenucleotide sequence of about 40 nucleotides of the fragment was shown tocorrectly encode residues 16 to 29 of mature Can f II (SEQ ID NO: 97).The missing residue in the amino acid sequence of a native protein atthe position 26 was found to be asparagine.

In order to generate a Can f II specific probe long enough (>100 bp) tobe used to screen a cDNA library, the 5′ and 3′ ends of the Can f IIcDNA were cloned using an anchored PCR technique (Roux, K. H. andDhanarajan, P., (1990) Biotechniques, 8: 48-57; Rafnar., T. et al.,(1991) J. Biol. Chem., 266: 1229-1236) (FIG. 15). Double stranded dogcDNA was synthesized as described above. The blunt double stranded cDNAwas than ligated to an anchor adapter AT/AL (FIG. 16; SEQ ID NO: 96 and102) thereby placing a known sequence at 5′ and 3′ ends of cDNAs (FIG.15A). In order to obtain the 5′ end of Can f II cDNA, a primer based onthe anchor sequence AP2 (FIG. 16) (SEQ ID NO: 95) was used as a 5′primer in conjunction with 3′ antisense primers, D2-1 (residues 22 to30) (FIGS. 13 and 16) (SEQ ID NO: 74), D2-2 (residues 17 to 25) (FIGS.13 and 16) (SEQ ID NO: 75) and D2-3 (residues 16 to 21) (FIGS. 13 and16) (SEQ ID NO: 76) based on known Can f II cDNA sequence obtained frominitial PCR. Sequential rounds of PCR (40×[92° C. 30 seconds/60° C. 1minute/75° C. 1 minute]) were carried out to amplify the 5′ end of theCan f II cDNA. In the 1° reaction, double stranded anchored cDNA wasused as a template along with 5′ AP2/3′ D2-1 primers; in the 2° reaction1/20th of the 1° reaction mixture was used with 5′ AP2/3′ D2-2 primers;in the 3° reaction 1/20th of 2° reaction mixture was used with AP2/D2-3primers). 1% agarose gel electrophoresis of the reaction productsrevealed the presence of a single band of ˜300 bp long. As expected fromthe position of primers (see FIG. 15), the 2° and 3° reactions productsmigrated faster than 1° reaction product. The amplified fragment fromthe 3° reaction was gel purified and subjected to DNA sequence analysis.It's authenticity as the 5′ end of the Can f II cDNA was confirmed bythe presence of the first N-terminal residues of mature Can f II protein(FIG. 15A, shaded residues). The 5′ portion of cDNA (FIG. 15A) (SEQ IDNO: 98) encoded part of the amino acid signal sequence which was notfound in mature Can f II. The 3′ portion of Can f II (FIG. 15B) (SEQ IDNO: 99) was synthesized in an analogous manner as the 5′ end except thatsingle stranded cDNA was used as a template and APA (FIG. 16) (SEQ IDNO: 100) was used as a 3′ primer and D2-4,(SEQ ID NO: 77), D2-5 (SEQ IDNO: 78) and D2-6 (SEQ ID NO: 79) (FIGS. 14 and 16) were used as internalprimers in PCR. Direct sequencing of the PCR product from the 3°reaction revealed the presence of 8 amino acids of the known Can f IIsequence followed by 8 amino acids downstream of the known sequence.

In order to clone the full length Can f II cDNA, a cDNA library wasprepared and screened using standard published procedures (Gubler andHoffman, Ausubel at al., supra). The lambda cDNA library was custom madeby Clontech Laboratories, Inc. as follows: the first strand cDNA wasprimed from poly(A) RNA by oligo d(T)15. The blunt ended double strandedcDNA was ligated to an Eco RI linker CCGGAATTCCGG (SEQ ID NO: 101),digested with EcoRI, size selected in order to obtain fragments largerthan 500 bp, and ligated into EcoRI cut and dephosphorylated vector λgt10. The DNA was then packaged into lambda particles, plated on C600-hfland C-600 E.Coli strains and the library titer was determined. Theunamplified library consisted of 1.53×106 independent clones (clearplaques on C-600 hfl host) which contained inserts ranging in size from0.6 kb to 3-4 kb. The average size of the insert as determined by PCRusing Clontech λgt10 primers was 1.2 kb. 100,000 clones were plated onC-600hfl host and screened using Can f II specific probe. Allmanipulations leading to the cloning and sequencing of Can f II cDNAwere done according to Protocols in Molecular Biology (Ausubel et al.,supra). A Can f II probe was obtained by PCR amplification of dog cDNAusing D2-9 (SEQ ID NO: 80) and D2-13 (SEQ ID NO: 83) primers (FIG. 16).The PCR product was then ³²P labeled by random priming. 20 positiveclones were plaque purified, phage DNA was extracted from individualclones, digested with EcoRI and subcloned into pUC18. The presence ofinserts was verified by digestion of the plasmid DNA with EcoRI and thenucleotide sequence of three individual clones was determined usingSequenase (United States Biochemicals) and AmpliTaq Cycle Sequencing kit(Perkin Elmer Cetus, Norwalk, Conn.) according to manufacturer'sinstructions. PCR cycle sequencing analysis served to resolve some DNAsequence ambiguities resulting most probably from the formation ofsecondary structures on GC-rich Can f II template. The sequencingstrategy is depicted in FIG. 17. Primers used in thesequencing/amplification included commercially available 16-mer ReverseSequencing Primer (−21) and 17-mer Sequencing Primer (−20) from NewEngland BioLabs as well as the Can f II specific primers listed in FIG.16.

The nucleotide sequence of the three clones revealed the presence of anopen reading frame which included 38 N-terminal amino acid residues(amino acids 1 to 38 on FIG. 13)(SEQ ID NO: 88) of mature Can f IIidentified earlier by protein sequencing and PCR sequencing of partialcDNA (see above). Sequencing strategy and features of three cDNA clones1a, 1c and 1j are shown on the FIG. 17. Clone 1c of 791 bp (SEQ ID NO:67) encodes the full length Can f II precursor protein (including signalsequence) and contains 5′ (bases 1 through 194) and 3′ (bases 738 trough791) untranslated regions. Clones 1a of 793 bp (SEQ ID NO: 69) and 1j of774 bp (SEQ ID NO: 71), encode precursor Can f II proteins in which partof the signal sequence is missing and contain 3′ untranslated regionswhich are longer then in 1c. The sequence alignment revealed apolymorphism among three clones (FIG. 18). The nucleotide sequence of lacontains one nucleotide substitution (C to T at the position 607) andone deletion (at the position 752) compared to 1c (FIG. 18). Thenucleotide sequence of 1j contains two nucleotide substitutions comparedto 1a and 1c at positions 347 (T to C) and 401 (G to T). In addition,the sequence of clones 1a and 1c differ significantly at their 5′ and 3′ends. The G to T substitution at the position 401 changes the predictedamino acid sequence of Can f II at residue 68 from glycine (GGC) tovaline (GTC). All other nucleotide changes do not alter the amino acidsequence of Can f II since they are either silent mutations or they lieoutside of the coding sequence of mature Can f II. The polymorphismamong the cDNA clones may reflect the expression of Can f II genes fromdifferent alleles. It may also represent a cloning artifact due to thereverse trancriptase mediated synthesis of cDNA which may introduceerrors (Holland et al., (1982) Science, 215: 1577-1585). For example,the purified HIV-1 reverse transcriptase was found to introducemisincorporations at a rate of 1/2000 to 1/4000 (Preston et al., (1988)Science, 242: 1168-1171). It is also possible that the formation ofsecondary structures on the GC-rich Can f II mRNA template may causepausing of reverse transcriptase or abnormal termination of thesynthesis.

The predicted sequence of Can f II protein shown in FIG. 18 (SEQ ID NO:68) contains a 19 amino acid signal sequence encoded by base 195 throughbase 251 of the cDNA shown in FIG. 18 (SEQ ID NO: 67). This signalsequence is not found in the mature Can f II protein which is encoded bybases 252 through 734. The methionine codon at the position −19 is trueinitiator methionine codon and not just an internal methionine residuebecause: 1) the predicted amino acid sequence of a signal peptide(residues −19 to −1) is highly similar and identical in length to thesignal sequences of proteins that are related to Can f II (see below);and 2) although another in-frame methionine codon is found 5′ of thepresumed initiatior metionine (position −53) it is unlikely to be truebecause the deduced amino acid sequence of a peptide starting at theresidue −53 is much longer than any known signal sequence and does notshow any similarity to any known signal sequence. The Can f II cDNAencodes a protein having a predicted molecular weight of 18.2 kDa, witha single potential N-linked glycosylation site. Because 1) no amino acidsignal was found at residue 25 during protein sequence analysis, and 2)the asparagine residue resides within a consensus sequence for N-linkedglycosylation (N26 K27 S28), these data strongly suggest that the N26residue is modified by N-linked glycosylation. N-linked glycosylationmay increase the molecular weight of the mature protein. The deducedamino acid sequence of the mature protein encoded by the nucleic acidsequence is identical to the known NH2-terminal and internal amino acidsequence determined by amino acid sequence analysis of purified Can f IIprotein conducted as described in Example 3.

The expression of Can f II in various tissues was studied using theNorthern blot technique. Poly (A) RNA or total RNA from various tissueswas separated by electrophoresis through a 1.5% agarose gel containing2.2 M formaldehyde. (Ausubel et al., supra). After electrophoresis, theseparated RNAs were transferred onto GeneScreen membrane (NEN).Transfer, hybridization with a 32P labeled Can f II probe [obtained byPCR mediated amplification using D2-9 (SEQ ID NO: 14) and D2-13 (SEQ IDNO: 83) (FIG. 16) primers] and washings of the filter were performedaccording to the manufacturer's instructions. It appeared that the Can fII probe hybridized specifically at high stringency to RNA from dogparotid gland and to RNA from tongue epithelial tissue (FIG. 20). It didnot hybridize to RNA from liver, or submaxiliary gland. Hybridizationwas observed to two bands of about 800 bp and 900 bp long, suggestingthat Can f II may be encoded by two mRNA species. It is unlikely thattwo RNAs are transcribed from two different genes since a Southern blotexperiment suggested that only a single copy Can f II geneis present inthe dog genome. The two mRNAs encoding Can f II may be due toalternative splicing or to degradation of the mRNA. The formerpossibility seems very likely since different splicing configurations inthe 3′ noncoding region has been described for proteins which aresimilar to Can f II (Clark et al., (1984) EMBO J., 3: 1045-1052, seealso below).

In order to infer the possible biological function of Can f II, itsamino acid sequence was compared to those in the GenBank, GenBankUpdate,EMBL, and EMBL Update sequence data bases using the NCBI BLAST networkservice (Altschul, S. F., et al., (1990) J. Mol Biol., 215: 403-410).Can f II precursor protein displayed high similarity to two groups ofrelated proteins: 1) Mouse Urinary Proteins (MUPs) (FIG. 21) (SEQ ID NO:90) and 2) urinary a-2-globulins of rat (A2U) (FIG. 21) (SEQ ID NO: 89).The sequences of MUPs and A2Us show them both to be members of thelipocalin protein family (Cavaggioni et al., (1987) FEBS Lett., 212:225-228). These are small proteins capable of binding hydrophobicmolecules with high affinity and selectivity. This family now containsover 20 different proteins, principally identified through sequencehomology (Flower et al., (1991) Biochim. Biophys. Res. Commun., 180:69-74). The function of MUP and A2U remains unclear, but it is proposedthat rodent urinary proteins are responsible for binding pheromones andtheir subsequent release from drying urine (Bocskei et al., (1992)Nature, 360: 186-188). They are synthesized at different levels in theliver and in the submaxillary, lachrymal, sublingual, parotid andmammary glands (Shahan et al., (1987) Mol. Cell. Biol., 7: 1947-1954).MUP IV for example, is expressed predominantly in the lachrymal andparotid glands, but not in liver (Shahan et al., supra). The amino acidsimilarity of Can f II, MUP and A2U as well as their pattern ofexpression may indicate that Can f II is canine homolog of lipocalins.Interestingly, immunologic and biochemical studies of MUPs andMUP-related proteins have shown that these proteins are important humanallergens (Lorusso et al., (1986) J. Allergy Clin. Immunol., 78: 928;Platts-Mills et al., (1987) J. Allergy Clin. Immunol., 79: 505; Gurka etal., (1989) J. Allergy Clin. Immunol., 83: 945-954).

EXAMPLE 5 Bacterial Expression of Can f I

Bacterial expression of Can f I was performed as follows. The vectorpET11d ΔHR His6 (Novagen, Madison, Wis.; modified at ImmuLogicPharmaceutical Corporation by J. P. Morgenstern) was modified forexpression of Can f I in E. coli, by removal of the internal EcoR Irestriction site (at residues E143F144) from the Can f I cDNA to beinserted in the vector. This modification was necessary since all DNAfragments in this vector are cloned in frame with the His6 NH2 terminalleader sequence at a mutual 5′ EcoR I site. Hence, EcoR I sites internalto the insert must be avoided. The pET11d ΔHR His6 vector also requiresthat inserts have a 3′ BamH I site. However, since restriction sitessuch as Bgl II and Bcl I are compatible with BamH I overhangs, theycould be placed at the 3′ end of the Can f I cDNA, avoiding the need tomutate the internal BamH I site. A cDNA encoding the mature Can f Iprotein had its internal EcoR I site removed, a unique EcoR I siteplaced at its 5′ end, and a Bgl II site placed at its 3′ end in a twostep PCR reaction (Ho et al., supra) using Pfu I DNA polymerase tominimize errors during amplification (FIG. 6). Two halves of the Can f IcDNA were amplified in primary PCR reactions (template: PCR fragmentfrom cycle sequencing, program: 40×[95° 30 seconds/60° C. 45 seconds/75°C. 45 seconds]) with the 5′ portion of the molecule being amplified withthe Start ex (SEQ ID NO:46 and SEQ ID NO:47)/EF antisense (SEQ ID NO:50and SEQ ID NO:48) primer pair and the 3′ portion amplified with thesense EF (SEQ ID NO:49 and SEQ ID NO:48)/TAG Bgl II (SEQ ID NO:51 andSEQ ID NO:52) primer pair. Both EF primers were designed to introduce apoint mutation in the EcoR I site at residues E143F144 of Can f I fromGAATTC to GAGTTC, which would maintain the E143 residue since glutamatecan be encoded by GAA or GAG codons.

Amplified DNA fragments of the expected size were isolated in gel slicesfrom a 0.6% low melt agarose gel, melted at 70° C., mixed and used astemplate in a secondary (2°) PCR reaction with Start ex and TAG Bgl IIprimers. Mutagenized regions bearing the E143F144 pair should hybridizein the initial stages of the reaction to link the 5′ and 3′ ends of theCan f I cDNA, while the extreme 5′ and 3′ primers should serve toamplify the intact mutagenized cDNA. The entire reaction wasphenol/chloroform extracted, EtOH precipitated, 70% EtOH washed, anddigested with EcoR I and Bgl II. A band of the expected size (˜450 bp)was isolated as a gel slice from a 0.6% low melt agarose gel, melted at70° C., ligated at room temperature to EcoR I/BamH I digested pET 11dΔHRHis 6 plasmid, and the ligation transformed into XL-1 bacteria(Stratagene). Miniprep analysis of a 3 ml culture of one of thetransformed colonies (using a Qiagen [Foster City, Calif.] plasmid minikit) by Eco RV digestion revealed the presence of an insert of theappropriate size within the expression vector. A 300 ml culture seededwith this colony was grown, plasmid, DNA extracted (Qiagen plasmid midikit) and subjected to DNA sequence analysis. The entire 453 bp insertwas shown to have the correct sequence for mature Can f I cDNA(including the mutated E143 codon from GAA to GAG), with the addition ofan in-frame His6 reporter group (SEQ ID NO:53) encoded at its 5′ end.This His6 reporter group was to be used in metal ion affinitypurification of the recombinant protein using NTA Ni++ chelating resin(Qiagen; Hochuli et al., supra).

A single colony of BL21 (DE3) pET 11d ΔHR His6Can f IdRI bacteria wasinoculated into a 2 ml brain heart infusion (BHI) culture (+200 μg/mlampicillin) and incubated at 37° C. until turbid but not saturated. Atthis point 6 μl was removed and added to 600 μl of BHI and mixed. 100 μlwas spread onto each of 6 BHI agar plates (+200 μg ampicillin) andincubated overnight at 37° C. The next morning the bacterial lawn wasscraped off of the plates, pooled and resuspended in 20 ml of BHI media,and then aliquoted 1 ml each into each of 18 500 ml BHI cultures (+200μg/ml ampicillin) in 2 liter Ehrlenmeyer flasks. Cultures were incubatedat 37° and shaken at 300 rpm until the A600 reached 1.0.Isopropyl-β-D-thiogalactopyranoside (IPTG) was than added to finalconcentration of 1 mM to induce expression of the T7 RNA polymerase genewhich would in turn induce expression of His6Can f I protein from thehybrid T7 gn10/lac 0 promoter. Expression was allowed to proceed for 2hours after which the bacteria were pelleted and resuspended in 6 Mguanidine hydrochloride (GuHCl), 100 mM NaPO4, 10 mM Tris, 100 mM2-mercaptoethanol pH 8.0. Extraction was carried out for 1 hour withvigorous shaking and terminated by pelleting of the insoluble materialat 10 K rpm in a JA-10 rotor (Beckman) for 1 hour. Supernatant wasremoved, and its pH adjusted to 8.0 before loading onto a 50 ml NTAagarose column that had been equilibrated in 6 M GuHCl, 100 mM NaPO4, 10mM Tris, pH 8.0. The column was washed by step gradient as follows: 1) 6M GuHCl, 100 mM NaPO4, 10 mM Tris, pH 8.0, 2) 8 M urea, 100 mM NaPO4, 10mM Tris, pH 8.0, 3) 8 M urea, 100 mM NaOAc, 10 mM Tris, pH 6.3 with eachwash proceeding until the A280 of the effluent from the column reachedbackground. Recombinant His6Can f I protein was eluted from the columnwith 8 M urea, 100 mM NaOAc, 10 mM Tris, pH 4.5. Yield of the pooledpeak fractions was ˜100 mg with a purity of ˜80% as determined bydensitometry of a sample of the material analyzed by SDS-PAGE.

E. coli transformed with the vector pET11d containing the nucleic acidencoding Can f I have been deposited with the ATCC at accession number69167.

EXAMPLE 6 Mammalian Expression of Can f I Protein

To produce a possibly glycosylated form of recombinant Can f I proteinexpessed in mammalian cells, Can f I expression was carried out asfollows. Full length Can f I protein (including the leader sequence notfound in the mature protein) when expressed in mammalian cells should beproperly folded, glycosylated, and secreted. Two systems for high leveltransient expression of recombinant Can f I were employed. First,transient expression of recombinant Can f I with a His6 reporter groupfused to its COOH terminus was performed in NIH 3T3 cells using the pJ7Ωexpression vector (Morgenstern, J. P. and Land, H., (1990) Nuc. AcidsRes., 18: 1068). pJ7Ω drives expression of genes inserted into itspolylinker to high levels during transient transfection from its SCMVIE94 promoter (Morgenstern and Land, supra).

A cDNA encompassing the entire Can f I coding sequence was amplifiedusing Pfu I mediated PCR of total dog parotid cDNA with the 5′ Kozakleader (SEQ ID NO:54)/3′ TAG Bgl II (SEQ ID NO:51 and SEQ ID NO:52)primer pair (see FIG. 7). The entire PCR reaction was phenol-chloroformextracted, EtOH precipitated, 70% EtOH washed and digested with Xho Iand Bgl II to generate correct overhangs for insertion into pJ7Ω. A bandof the expected size (˜600 bp) to encode the entire Can f I cDNA wasisolated as a gel slice on a 0.6% low melt agarose gel, melted at 70° C.and ligated to Sal I/Bgl II digested pJ7Ω at room temperature (see FIG.7). The ligation was transformed into competent XL-I Blue E. coli andpositive colonies selected on ampicillin (200 μg/ml) dishes. DNAsequence analysis of the 5′ and 3′ ends of inserts was performed onplasmid obtained from 3 ml cultures of two colonies (using a Qiagenplasmid mini kit). Both plasmids had inserts with the correct sequenceof the 5′ and 3′ ends of full length Can f I.

Next, to aid in purification of recombinant Can f I protein produced inmammalian cells (Jankecht, R., et al., (1991) Proc. Natl. Acad. Sci.USA, 88: 8972-8976), a His6 reporter group was to be fused at its COOHterminus. This was accomplished by excising the DNA fragment encodingthe COOH terminus of Can f I as an EcoRI-Bgl II fragment and exchangingit with an EcoR I-Bgl II fragment encoding the COOH terminus of theprotein that had been modified with the addition 6 histidines (FIG. 8).The COOH terminal His6 DNA fragment was generated by PCR of overlappingsynthetic oligonucleotides as follows: a sense oligonucleotide (SEQ IDNO:56) encoding residues E123 to Q148 of the mature Can f I protein (SEQID NO:55); Sense 3′ His6 link (SEQ ID NO:57); an antisenseoligonucleotide encoding residues E141 to Q148/a His 6 tract/stop codon(SEQ ID NO:50 and SEQ ID NO:59); and 3′ His6 TAG BglII (SEQ ID NO:60),were synthesized and purified by OPC column chromatography (AppliedBiosystems, Foster City, Calif.). In addition, smaller primers composedof the first 24 nucleotides of the aforementioned oligonucleotides, 5′His6 link and 3′ His6 link, were also synthesized. Linking andamplifying the two long oligonucleotides to generate the EcoR I-Bgl IIDNA fragment encoding the Can f I COOH terminus-His6 fusion wasperformed by PCR. 10 pmoles of each large oligonucleotide were used assubstrate in Pfu I mediated PCR with 1 μM primers using the program40×(95° C. 30 seconds/60° C. 45 seconds/75° C. 30 seconds). The entirePCR reaction was phenol-chloroform extracted, EtOH precipitated, 70%EtOH washed and digested with EcoR I and Bgl II to generate correctoverhangs for insertion into pJ7Ω Can f I. A band of the expected size(˜110 bp) to encode the Can f I COOH terminus/His6 fusion was isolatedas a gel slice on a 2.0% NuSieve agarose gel, melted at 70° C. andligated to EcoR I/Bgl II digested pJ7Ω Can f I at room temperature. Theligation was transformed into competent XL-1 Blue E. coli and positivecolonies selected on ampicillin (200 μg/ml) dishes. Plasmid was isolatedfrom 4 cultures inoculated with different colonies and subjected to DNAsequence analysis at the 3′ end of the insert. Clone #3 contained theexpected His6 residues linked in frame to the COOH terminus of Can f I,so a large scale grow of this culture was undertaken to obtain largequantities of the pJ7Ω Can f I His6 plasmid for transfection. A oneliter culture was amplified in 15 μM chloramphenicol once A600 reached0.6. 800 μg of plasmid were isolated after alkaline lysis and twosuccessive rounds of CsCl banding (Sambrook et al., supra).

Ten plates of NIH 3T3 cells were seeded at a density of 1.7×10⁶ cellsper 15 cm tissue culture dish, and the following morning, subjected tocalcium phosphate transfection with 20 μg/dish of pJ7Ω Can f I His6plasmid (Parker, B. A., and Stark, G. R., (1979) J. Virol., 31:360-369). 48 hours post transfection, supernatant was pooled from thedishes, filtered through a 0.45μ unit (Costar), and brought to aconcentration of 1 μM imidazole, with the addition of proteaseinhibitors 1 mM PMSF, 1 μg/ml, pepstatin 1 μg/ml soybean trypsininhibitor, and 1 μg/ml leupeptin. Metal ion affinity purification of theCan f I His6 protein was achieved by loading the supernatant onto a 2 mlNTA agarose (Qiagen) column that had been equilibrated in 1×PBS, 1 mMimidazole, 1 mM PMSF, 1 μg/ml pepstatin, 1 μg/ml soybean trypsininhibitor, and 1 μg/ml leupeptin. Non-specifically bound proteins werewashed off the column with 10 column volumes of 1×PBS, 20 mM imidazole,1 mM PMSF, 1 μg/ml pepstatin, 1 μg/ml soybean trypsin inhibitor, and 1μg/ml leupeptin. Can f I His6 protein was specifically eluted from thecolumn in 1×PBS, 80 mM imidazole, 1 mM PMSF, 1 μg/ml pepstatin, 1 μg/mlsoybean trypsin inhibitor, and 1 μg/ml leupeptin (Hoffmann, A. andRoeder, R. G., (1991) Nuc. Acids. Res., 19: 6337-6338, and Janknecht etal., supra). Aliquots of the eluted fractions were analyzed by 12% SDSPAGE. Coomasie blue staining of the gel revealed three major bands ofmolecular weight ˜70 kDa, 45 KDa, and 25 KDa. Since the molecular weightof native immunoaffinity purified Can f I is 25 KDa (Schou et al., supraand de Groot et al., supra) it was suspected that the smallest band onthe SDS gel was recombinant Can f I His6.

EXAMPLE 7 Direct Binding of Human IgE to Recombinant Can f I by ELISAand Western Blot

An ELISA plate (IMMULON II Dynatech, Chantilly, Va.) was coated withbacterially expressed recombinant Can f II, (rCan f I) at 0.5 μg/well inPBS-Tween and incubated overnight at 4° C. The coating antigen wasremoved and the wells were blocked with 0.5% gelatin in PBS, 200 μl/wellfor two hours at room temperature. Plasma from a skin test positive dogallergic patient, #901, was serially diluted with PBS-Tween and 100 μlwas added per well and incubated overnight at 4° C. (the plasmadilutions were tested in duplicate). The second antibody (biotinylatedgoat anti-human IgE 1:1000, Kirkegaard & Perry Laboratories) was addedat 100 μl/well for one hour at room temperature. This solution wasremoved and streptavidin-HRPO at 1:10,000, (Southern BiotecnologyAssociates, Inc. Birmingham, Ala.) was added for one hour at roomtemperature. TMB Membrane Peroxidase Substrate system (Kirkegaard &Perry Laboratories) was freshly mixed and added at 100 μl. The color wasallowed to develop for 2-5 minutes. The reaction was stopped by theaddition of 100 μl/well of 1 M phosphoric acid. The plate was read on aMircoplate EL 310 Autoreader (Biotek Instruments, Winooski, Vt.) with a450 nm filter. The absorbance levels of duplicate wells were averaged.The graphed results are shown in FIG. 11. The data shows that patient#901 has a high level of anti-Can f specific IgE such that at 1/162dilution (the highest plasma dilutin used) the binding level to therecombinant Can f I is still two-fold above background. A known negativepatient (#250) was also tested and shown to be negative by this assay.

Western Blot analysis of four different protein preparations aspotential sources Can f I was performed. The four differentprepparations used for Western blotting were: dog hair extract, dogsaliva, bacterially expressed rCan f I (used for the ELISA) and rCan f Ias expressed in a mammalian cell culture system. These preparations wereloaded on a 15% acrylamide SDS-PAGE (lanes 1-4, respectively) at 5μg/lane. The protein concentrations were based on the Bicinchoninic acid(BCA) assay (Pierce, Rockford, Ill.). Following electrophoresis, theproteins were transferred to nitrocellulose and stained with India Ink.The nitrocellulose sections were blocked by incubation in Tween solutionwith 1% milk/1% BSA for 30 minutes room temperature, then probed withpatient #901 plasma or negative control patient #250 at a 1:20 dilutionin tween mild solution. This first antibody incubation was carried outovernight at room temperature. Biotinylated goat anti-human IgE (KPL)was used as the second antibody at a 1:5000 dilution for a two hourincubation. Streptavidin-HRPO (1:20,000 dilution) and the ECL WesternBlot Detection system (Amersham, Arlington Heights, Ill.) were used fordetection by chemiluminescence. A 20 second exposure was performed andthe film developed. The results from this assay show no recognition ofthe protein preparations by patient #250 IgE. The IgE from dog allergicpatient #901 shows distinct binding to Can f I proteins in the salivaand the bacterially expressed recombinant Can f I (lanes 2 and 3, FIG.12). The sizes of the protein forms are different between the twopreparations and this is due to fact that the native Can f I proteinfound in dog saliva is glycosylated and runs with an apparent molecularweight of 28,000 daltons whereas the recombinant form from bacteria hasno carbohydrate modification. The binding of IgE from serum of patients#901 to the mammalian expressed rCan f I is extremely faint and atpresent is only suggestive of positive expression. The full lengthbacterially produced form, lane 3, has an apparent molecular weight of18,000 daltons and the larger IgE binding proteins in both lanes 2 and 3are most likely dimeric structures of the lower molecular weightproteins.

EXAMPLE 8 Bacterial Expression of Can f II

In an attempt to readily produce large amounts of pure recombinant Can fII protein, expression of Can f II in bacteria was carried out asfollows. Full length cDNA encoding mature Can f II protein was obtainedby amplification of the molecule from total parotid cDNA in PCR usingD2-3pet (SEQ ID NO: 103) and D2-5pet (SEQ ID NO: 104) primer pair (FIG.13). Primers were designed to introduce EcoRI and BamHI restrictionsites at 5′ and 3′ of cDNA molecule respectively. The pET11d ΔHR His6vector requires that inserts have 5′ EcoRI site and 3′ BamHI site.

Amplified DNA fragment of the expected size was purified byelectrophpresis in low melting agarose and ligated at room temperatureto EcoRI/BamHI digested pET11d ΔHR His6 plasmid and the ligation mixturewas used to transform XL-1 Blue bacteria (Stratagene). Miniprep analysisof several transformed colonies (using Qiagen [Foster City, Calif.]plasmid mini kit) revealed the presence of an insert of the appropriatesize within the expression vector. A 300 ml culture inoculated with onecolony was grown, plasmid DNA extracted and subjected to sequenceanalysis. The entire 486 bp insert was shown to have the correctsequence for the mature Can f II cDNA with the addition of a reportergroup encoded at the 5′ end. This His6 reporter group was to be used inmetal ion affinity purification of the recombinant protein using NTANi++ chelating resin (Qiagen). A single colony of BL21(DE3) pET 11d ΔHRHis6Can f II bacteria was inoculated into a 2 ml brain heart infusion(BHI) culture (+200 μg/ml ampicillin) and incubated at 37° C. untilturbid but not saturated. At this point 6 μl was removed and added to600 μl of BHI and mixed. 100 μl was spread onto each of 6 BHI agarplates (+200 μg ampicillin) and incubated overnight at 37° C. The nextmorning the bacterial lawn was scraped off of the the plates, pooled andresuspended in 20 ml of BHI media, and then aliquoted 1 ml each intoeach of 18 500 ml BHI cultures (+200 μg/ml ampicillin) in 2 literEhrlenmeyer flasks. Cultures were incubated at 37° C. and shaken at 300rpm until the A600 reached 1.0. Isopropyl-β-D-thiogalactopyranoside(IPTG) was than added to 1 mM to induce expression of the T7 RNApolymerase gene which would in turn induce expression of His6Can f IIprotein from the hybrid T7 gn10/lac O promoter.

Expression was allowed to proceed for 2 hours after which the bacteriawere pelleted and resuspended in 6 M guanidine hydrochloride (GuHCl),100 mM NaPO4, 10 mM Tris, 100 mM 2-mercaptoethanol pH 8.0. Extractionwas carried out for 1 hour with vigorous shaking and terminated bypelleting of the insoluble material at 10 K rpm in a JA-10 rotor(Beckman) for 1 hour. Supernatant was removed, and its pH adjusted to8.0 before loading onto a 50 ml NTA agarose column that had beenequilibrated in 6 M GuHCl, 100 mM NaPO4, 10 mM Tris, pH 8.0. The columnwas washed by step gradient as follows: 1) 6 M GuHCl, 100 mM NaPO4, 10mM Tris, pH 8.0, 2) 8 M urea, 100 mM NaPO4, 10 mM Tris, pH 8.0, 3) 8 Murea, 100 mM NaOAc, 10 mM Tris, pH 6.3 with each wash proceeding untilthe A280 of the effluent from the column reached background. RecombinantHis6Can f II protein was eluted from the column with 8 M urea, 100 mMNaOAc, 10 mM Tris, pH 4.5. Yield of the pooled peak fractions was ˜100mg with a purity of ˜80% as determined by densitometry of a sample ofthe material analyzed by SDS-PAGE.

E. coli transformed with the vector pET11d containing the nucleic acidencoding Can f II have been deposited with the ATCC at accession number69167.

EXAMPLE 9 Direct Binding of Human IgE to Native and Recombinant Can f II

Plasma samples from 14 dog-allergic patients (skin test 4+) were assayedfor IgE binding to native Can f II and rCan f II. An ELISA plate(Immulon II Dynatech, Chantilly, Va.) was coated with native andbacterially expressed recombinant Can f II at 0.5 μg/well in PBS-Tweenand incubated overnight at 4° C. The coating antigen was removed and thewells were blocked with 0.5% gelatin in PBS, 200 μl/well for two hoursat room temperatre. Binding of human IgE to the coating antigen wasdetected using biotinylated goat-anti-human IgE, streptavidin linked toperoxidase and TMB substrate. Reactions were read on a plate reader at450 nm (A450). Of 14 plasma samples tested for IgE to native Can f II, 5contained detectable antibody binding (FIG. 22A), with plasma frompatients #901 and #227 containg the highest levels. Similarly, of 23plasma samples tested for IgE to recombinant, bacterially expressed Canf II, several contained detectable antibody binding (FIGS. 22B and C).

EXAMPLE 10 Can f I Human T Cell Proliferation Analysis

To identify peptides having Can f I T cell stimulating activity severalpeptides derived from Can f I were produced and cultured with human Tcell lines primed with recombinant Can f I protein and the responseswere determined by standard T cell proliferation assays. A set ofpeptides derived from Can f I (Construct I (SEQ ID NO: 105), Construct 2(SEQ ID NO:106), and Construct 3 (SEQ ID NO:107)), each representing aportion of the Can f I protein were used in proliferation assays. Inaddition, the assays included two peptides (A0095 (SEQ ID NO:108), aminoacids 7 through 19; and A0096 (SEQ ID NO:109); amino acids 42 through54) which were selected as containing potential T cell epitopes usingthe algorithm described in Hill et al., Journal of Immunology 147:184-197.

Constructs 1, 2 and 3 were produced by expressing and purifying portionsof the Can f I protein in E. coli using standard techniques. DNAfragments encoding Construct I (amino acids 1 through 65 of Can f I) andConstruct 2 (amino acids 56 through 108) were obtained by amplificationfrom a plasmid pET11dΔ HRHis6Can f IΔ containing full length Can f IcDNA and were subcloned into an EcoRI/BamHI site of pET11d vectorcontaining a His6 reporter sequence. A DNA fragment encoding Construct 3(amino acids 90 through 148) was amplified from the same plasmid andsubcloned into an EcoRI site of pET11d vector. Recombinant proteins wereaffinity purified on a NTA Ni⁺⁺ chelating resin (Qiagen) according topublished protocol.

In order to perform in vitro assays of human T cell proliferativeresponse to Can f I and the five Can f I peptides described above, wholeblood obtained from subjects allergic to Can f I in a skin prick testwas passed through a Lymphocyte Separation Media (LSM) to removeplatelets, red blood cells and granulocytes. The resulting peripheralblood mononuclear cells (PBMC) were stimulated with 50 μg/ml ofrecombinant Can f I produced as described in Example 2 for 6 days inRPMI 1640 medium supplemented with 5% heat inactivated human AB serum, 2mM L-glutamine, 10 mM HEPES, 50 μM 2-mercapto-ethanol and 100 U/mlpenicillin and streptomycin.

A second separation LSM was performed to remove high density cell debrisand dead cells. The resulting PBMC cells were allowed to proliferate for12-18 days. During this time the medium was supplemented with additionsof recombinant IL2 (5units/ml) and IL4 (5 units/ml.).

When the lymphocytes reached a point of rest (determined such that anovernight pulse of 20,000 cells with ³H thymidine was within the2000-4000 CPM range), the cells were restimulated for analysis insecondary proliferation assays. Secondary T cell proliferation assaysincluded 2×10⁴ T cells/well, 5×10⁴ PBMC/well (irradiated with 3500 rads)as antigen presenting cells. Antigens were assayed in duplicate ortriplicate wells at the following concentrations:

rCan f I: 4, 20 and 100 μg/ml.

Peptides: 3, 15, and 75 μg/ml.

Dog extract: 3, 15 and 75 μg of protein/ml. The concentration of Can f Iin this preparation dog extract is unknown.

Constructs: initially at a single concentration of 20 μg/ml, then at 3,15, and 75 μg/ml.

Constructs were insoluble at 75 μg/ml.

PHA was added at 1 μg/ml to indicate nonspecific activation ability.Tetanus toxoid, an irrelevant antigen, was added at dilutions of 1:2000,1:4000 and 1:8000 to indicate T cell specificity.

After 3 days of culture under conditions of secondary assay 1 μCi of ³Hthymidine was added to each culture well for overnight incubation.Cultures were harvested on glass fiber filters and ³H thymidineincorporation was measured by β scintillation counting. Stimulationindices which measure the strength of a T cell response to a peptidewere calculated by dividing ³H thymidine uptake of treated cultures by³H thymidine uptake of untreated medium controls.

The results of the secondary T cell proliferation assays are shown inFIGS. 24-25. FIG. 24 graphically compares the stimulation indices ofindividual subjects to rCan f I, peptides A0095-A0096 and Constructs1-3. This comparison indicates that significant areas of T cellreactivity in the Can f I protein are found at all three parts of theprotein, as shown by the substantial stimulation indices of Constructs1-3 which, together, encompass the entire Can f I protein sequence.

Positivity indices for the peptides shown in FIG. 10 were calculated bymultiplying the mean T cell stimulation index (FIG. 25) by the percentof the tested individuals who had a positive response or a T cellstimulation index of at least two. The percentage of positive respondersfor each tested peptide were as follows: rCan I: 89%, A0095: 43%, A0096:43%, Construct 1: 64%, Construct 2: 73%, Construct 3: 82%. Comparison ofpositivity indices (FIG. 10) which measures both the strength of a Tcell response to a peptide (S.I.) and the frequency of a T cell responseto a peptide in a population of dog dander allergen sensitiveindividuals indicates that both N terminal (amino acids 1 through 65)and C terminal (amino acids 90 through 108) ends of the Can f I proteincontain a number of T cell epitopes.

EQUIVALENTS

Although the invention has been described with reference to itspreferred embodiments, other embodiments can achieve the same results.Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, numerous equivalents to the specificembodiments described herein. Such equivalents are considered to bewithin the scope of this invention and are encompassed by the followingclaims.

1. A method of detecting sensitivity to dog dander allergen in a mammalsensitive to dog dander allergen, comprising administering to saidmammal an isolated Can f II dog dander allergen, wherein said allergenis encoded by the nucleotide sequence of SEQ ID NO: 67, detecting thepresence of an IgE mediated immune response in said mammal, andcorrelating the response with the presence of said dog dander allergenin the mammal.
 2. A method of detecting sensitivity to dog danderallergen in a mammal sensitive to dog dander allergen, comprisingadministering to said mammal an isolated Can f II dog dander allergen,wherein said allergen is encoded by a nucleic acid comprising anucleotide sequence which hybridizes under conditions of high stringencyto a complementary strand of the nucleotide sequence of SEQ ID NO: 67,detecting the presence of an IgE mediated immune response in saidmammal, and correlating the response with the presence of said dogdander allergen in the mammal, wherein the hybridization conditionsinclude 0.2× sodium chloride/sodium citrate (SSC) at about 50-65°C.
 3. Amethod of detecting sensitivity to dog dander allergen in a mammalsensitive to dog dander allergen, comprising administering to saidmammal an isolated antigenic peptide comprising at least one T cellepitope of a Can f II dog dander allergen, wherein said peptide isencoded by a portion of the nucleotide sequence of SEQ ID NO: 67,detecting the presence of an IgE mediated immune response in saidmammal, and correlating the response with the presence of said peptidein the mammal.